Methods of decreasing cell adhesion

ABSTRACT

The present invention provides substantially pure integrins containing a novel β subunit designated as β 6 . The novel β 6  subunit forms heterodimers with α V  and α F . Methods of controlling cell adhesion using the β 6 -containing integrins are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patent application Ser. No. 09/591,543, filed Jun. 8, 2000, now abandoned, which is a divisional application of U.S. patent application Ser. No. 08/938,085, filed Sep. 26, 1997, now U.S. Pat. No. 6,339,148, which is a divisional application of U.S. patent application Ser. No. 07/728,215, filed Jul. 11, 1991, now U.S. Pat. No. 5,962,643, each of which is incorporated herein by references.

This work was supported in part by research grants HL/AL 33259, CA-47541 and CA-47858 from the National Institutes of Health. The U.S. Government has rights in the invention.

BACKGROUND ART

This invention relates to receptors for adhesion peptides, and more specifically to a novel receptor subunit having affinity for extracellular matrix molecules.

Multicellular organisms, such as man, have some 10¹⁴ cells which can be divided into a minimum of fifty different types, such as blood cells and nerve cells. During the course of growth and development, cells adhere to other cells, or to extracellular materials, in specific and orderly ways. Such cell adhesion mechanisms appear to be of importance in mediating patterns of cellular growth, migration and differentiation, whereby cells develop specialized characteristics so as to function as, for example, muscle cells or liver cells. Cell adhesion mechanisms are also implicated in dedifferentiation and invasion, notably where cells lose their specialized forms and become metastasizing cancer cells.

The mechanisms underlying the interactions of cells with one another and with extracellular matrices are not fully understood, but it is thought that they are mediated by cell surface receptors which specifically recognize and bind to a cognate ligand on the surface of cells or in the extracellular matrix.

The adhesion of cells to extracellular matrices and their migration on the matrices is mediated in many cases by the binding of a cell surface receptor to an Arg-Gly-Asp containing sequence in the matrix protein, as reviewed in Ruoslahti and Pierschbacher, Science 238:491-497 (1987). The Arg-Gly-Asp sequence is a cell attachment site at least in fibronectin, vitronectin, fibrinogen von Willibrand, thrombopondin, osteopontin, and possibly various collagens, laminin and tenascin. Despite the similarity of their cell attachment sites, these proteins can be recognized individually by their interactions with specific receptors.

The integrins are a large family of cell surface glycoproteins that mediate cell-to-cell and cell-to-matrix adhesion as described, for example, in the Ruoslahti and Pierschbacher article cited above. All known members of this family of adhesion receptors are heterodimers consisting of an α and a β subunit noncovalently bound to each other. When the integrin family was first identified, integrins were grouped into three subfamilies based on the three β subunits that were initially recognized (β₁, β₂ and β₃). Over the past few years, the primary structures of three integrin β subunits from mammalian cells and one from Drosophila have been deduced from cDNA.

Each α subunit was thought to associate uniquely with a single β subunit. Eleven distinct a subunits have thus far been described. As new integrins have been identified, however, it has become clear that this grouping is not entirely satisfactory, since there are clearly more than three β subunits and since some α subunits can associate with more than one β subunit as described, for example, in Sonnenberg et al., J. Biol. Chem. 265:14030-14038 (1988).

Because of the importance of integrins in mediating critical aspects of both normal and abnormal cell processes, a need exists to identify and characterize different integrins. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to a substantially purified β subunit of an integrin cell surface receptor designated as β₆. The amino acid sequence of human β₆ (SEQ ID NO:27) is provided in FIG. 3.

The present invention also relates to amino acid fragments specific to β₆ that have a variety of uses. The invention further relates to vectors having a gene encoding such fragments. Host cells containing such vectors are also provided. The nucleic acids encoding β₆ as well as nucleic acids that specifically hybridize with the nucleic acids encoding β₆ sequences are other aspects of the present invention.

In a further aspect, the present invention relates to a substantially purified integrin comprising β₆ bound to an α subunit, particularly α_(V) or α_(F). Methods of blocking the attachment of the β₆-containing integrins to its ligand and of detecting the binding of such integrins to its ligand are also provided.

The present invention also relates to methods of increasing or decreasing cell adhesion in cells expressing a β₆-containing integrin by overexpressing the integrin or by binding the integrin with a ligand, such as vitronectin.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the design of consensus PCR primers (SEQ ID NOS:1-5, 7 and 8) (β₂ human nucleic acids=SEQ ID NOS:10, 14, 18 and 22; corresponding β₂ human amino acids=SEQ ID NOS:50, 51, 54 and 55; β₃ human nucleic acids=SEQ ID NOS:11, 15, 19 and 23; corresponding β₃ human amino acids=SEQ ID NOS:52, 53, 56 and 57; β₁ human nucleic acids=SEQ ID NOS: 12, 16, 20 and 24; corresponding β₁ human amino acids=SEQ ID NOS:50, 53, 58 and 59; β₁ chicken nucleic acids=SEQ ID NOS:13, 17, 21 and 25; corresponding β₁ chicken amino acids=SEQ ID NOS:50, 53, 60 and 59; β₆ guinea pig sequence from position 219=SEQ ID NO:6; corresponding β₆ guinea pig amino acids=SEQ ID NO:61; β₆ guinea pig sequence from position 1325=SEQ ID NO:8; corresponding β₈ guinea pig amino acids=SEQ ID NO:62).

FIG. 2 shows a map of sequencing strategy.

FIGS. 3A-3C show the nucleotide sequence and amino acid translation for human (H) (SEQ ID NOS:26 and 27) and guinea pig (GP) (SEQ ID NOS:28 and 29).

FIGS 4A-4C show the alignment of human β₆ (SEQ ID NO:27) with, four previously reported integrin β subunits (human β₁=SEQ ID NO:30; human β₂=SEQ ID NO:31; human β₃=SEQ ID NO:32; Drodophila β_(myo)=SEQ ID NO:33).

FIGS. 4A-4B show the nucleotide sequence and amino acid translation for human (H) (SEQ ID NOS:26 and 27) and guinea pig (GP) (SEQ ID NOS:28 and 29).

FIGS. 1A-1B show the alignment of human β₆ (SEQ ID NO:27) with, four previously reported integrin β subunits (human β₁=SEQ ID NO:30; human β₂=SEQ ID NO:31; human β₃=SEQ ID NO:32; Drosophila β_(myo)=SEQ ID NO:33).

FIGS. 5A-5B show the alignment of partial nucleotide and amino acid sequences from human (H) and guinea pig (GP) β(human (β_(1H))=SEQ ID NOS:34 and 35; guinea pig (β_(1GP))=SEQ ID NOS:36 and 37, respectively), β₃ (human (β_(3H)=SEQ ID NOS:38 and 39; β₃ (human (β_(3H))=SEQ ID NOS:39 and 40, respectively), and β₆ (human (β_(6H))=SEQ ID NOS:42 and 43; guinea pig (β_(6GP))=SEQ ID NOS:44 and 45, respectively) for the region just downstream from the β3F primer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition of matter relating to a novel, substantantially purified integring βsubunit, referred to herein as β₆. The amino acid sequence of β₆ for humans (SEQ ID NO;27) and for guinea pig (SEQ ID NO:29) are also provided and are shown in FIG. 3.

By “substantially purified” is meant substantially free of contaminants normally associated with a native or natural enviroment.

By “β₆” is meant a polypeptide having substantially the same amino acid sequence and binding functions of the polypeptides encoded by the sequences set forth in FIG. 3 for human (SEQ ID NO:26) and guinea pig (SEQ ID NO:28) β₆. Thus, modified amino acid sequences that do not substantially destroy the functions and retain the essential sequence of β₆ are included within the definition of β₆. Amino acid sequences, such as the sequence for ⊕₁ (SEQ ID NO:30), β2 (SEQ ID NO:31), and β₃ (SEQ ID NO:32), having less than 50% homology with the sequence of β₆, are not substantially the same sequence and, therefore, do not fall within the definition of β₆. Given the amino acid sequences set forth herein, additions, deletions or substitutions can be made and tested to determine their effect on the function of β₆. In addition, one skilled in the art would recognize that certain amino acids, such as the conserved cystines, for example, can be modified to alter a binding function of β₆.

Amino acids are identified herein by the standard one-letter abbreviations, as follows:

Amino Acid Symbol Alanine A Asparagine N Aspartic acid D Arginine R Cysteine C Glutamine Q Glutamic acid E Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

Based on its amino acid sequence, the β subunit of the present invention is clearly different from β₁, β₂, β₃ and other β subunits that have recently been discovered. For example, the 11-amino acid carboxyl-terminal extension on β₆ distinguishes it from β₁, β₂, and β₃. The short cytoplasmic tails of β₁, β₂, and β₃ are thought to be sites of interaction with the cytoskeleton and regions for the transduction of signals initiated by interactions of the large extracellular domains with ligands. These cytoplasmic tails may also be targets for regulation of integrin function. The distinctive 11-amino acid cytoplasmic tail of β₆ indicates that its regulation or pathways for signal transduction may be different from those of β₁, β₂ and β₃.

In addition to β₁, β₂ and β₃, recent studies have suggested the existence of as many as five other integrin β subunits. A β subunit with a molecular weight of approximately 210,000 (β₄) has been found associated with the integrin α subunit “α₆ ¹” in colon carcinoma cells and in a variety of other tumor cells of epithelial origin as described, for example, in Kajiji et al., EMBO J., 8:673-680 (1989). On the basis of its high molecular weight, 210,000 compared with the predicted size of 106,000 of the subject novel protein, and on the basis of its clearly different amino-terminal sequence, it is apparent that β₄ is not the same as the subject polypeptide.

Another β subunit, originally called β_(X) was identified in epithelial-derived tumor cells in association with the integrin α subunit α_(V) as described, for example, in Cheresh et al., Cell 57:59-69 (1989). This β subunit, having a distinctive amino-terminal sequence, was recently renamed β₅. Based on recent studies of purified preparations, β₅ clearly differs from the β subunit of the present invention. Because the β subunit described in the present report is distinct from each of the five β subunits for which sequence information is available, it has been designated as β₆.

The existence of two other integrin β subunits has been inferred from the identification of unique proteins after immunoprecipitation of surface-labeled cell lysates with antibodies to known α subunits. One of these novel proteins, called β_(S) was found in association with α_(V) in the human osteosarcoma cell line MG-63, in the fibroblast cell line AF1523, and in human endothelial cells as described, for example, in Freed et al., EMBO J. 8:2955-2965 (1989). This subunit is also different from μ₆ since β_(S) is expressed in MG-63 cells while β6 is not expressed in these cells as shown in Table 1.

The other novel integrin β subunit identified by co-immunoprecipitation of known α subunits, β_(P), is a protein of about M_(r) 95,000 that is found to be associated with α₄, an α subunit first found as part of the lymphocyte homing receptor VLA-4 as described, for example, in Holzmann et al., Cell 45:37-46 (1989). This subunit is also distinct from β₆ since β_(P) is expressed in lymphocytes while β₆ is not expressed in lymphocytes as shown in Table 1.

TABLE 1 Distribution of β_(δ) Type Results Source Cell Lines: FG-2 Pancreatic + Kajiji et al., EMBO J. 3:673- 80 (1989) Panc I Pancreatic − Dr. Metzgar, Duke U., N.C. Colo-396 Colon CA + Dr. L. Walker, Cytel, San Diego, CA UCLA P3 Lung CA + Dr. L. Walker, Cytel, San Diego, CA HeLa Cervical − ATCC #CCL-2 Jar Chorio CA + ATCC #HTB 36 HT 1080 Fibrosarcoma − ATCC #CCL 121 U 937 Monocytoid − ATCC #CRL 1593 M 21 Melanoma − Dr. R. Reisfeld, Scripps Clinic & Research Foundation, La Jolla, CA B 16 Melanoma − Dr. R. Reisfeld Scripps Clinic & Research Foundation, La Jolla, CA MG 63 Osteosarcoma − ATCC #CRL 1427 Tissues: Cervix + Aortic Endothelium − Leukocytes −

The invention also provides an integrin comprising β₆ bound to an α subunit. β₆, consistent with recent findings of other β subunits, can associate with a variety of α subunits to form a functional integrin. In one embodiment, β₆ associates with α_(V). In another embodiment, β₆ associates with another α subunit referred to herein as α_(F). The α_(V)β₆ integrin, as well as other integrins containing β₆, can bind molecules, for example extracellular matrix molecules. Such molecules are referred to herein as ligands. In a specific embodiment, certain β₆-containing integrins can bind Arg-Gly-Asp-containing polypeptide's such as vitronectin or fibronectin. The binding of β₆-containing integrins to various ligands can be determined according to procedures known in the art and as described for example, in Ruoslahti and Pierschbacher, Science 238:491-497 (1987).

The invention also provides an amino acid fragment specific to β₆. Since β₆ is a novel molecule, it contains many fragments which are specific for this β subunit. Fragments specific to β₆ contain sequences having less than 50% homology with sequences of other known integrin β subunit fragments. These fragments are necessarily of sufficient length to be distinguishable from known fragments and, therefore, are “specific for β₆.” The amino acid sequence of such fragments can readily be determined by referring to the figures which identify the β₆ amino acid sequences. These fragments also retain the binding function of the β₆ subunit and can therefore be used, for example, as immunogens to prepare reagents specific for β₆ or as an indicator to detect the novel β₆-containing integrin of the present invention. One skilled in the art would know of other uses for such fragments.

The invention also provides a reagent having specificity for an amino acid sequence specific for β₆. Since β₆ is a novel protein with at least 50% amino acid differences over related β subunits, one skilled in the art could readily make reagents, such as antibodies, which are specifically reactive with amino acid sequences specific for β₆ and thereby immunologically distinguish β₆ from other molecules. Various methods of making such antibodies are well established and are described, for example, in Antibodies, A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory 1988, pp. 139-283 and Huse et al., Science 24:1275-1280 (1988).

The invention also provides nucleic acids which encode β₆. Examples of such sequences are set forth in FIG. 3 (SEQ ID NOS:26 and 28). Following standard methods as described, for example, in Maniatis et al., Molecular Cloning, Cold Spring Harbor (1982), nucleic acid sequences can be cloned into the appropriate expression vector. The vector can then be inserted into a host, which will then be capable of expressing recombinant proteins. Thus, the invention also relates to vectors containing nucleic acids encoding such sequences and to hosts containing these vectors.

The sequences set forth in FIG. 3 (SEQ ID NOS:26 and 28) also provide nucleic acids that can be used as probes for diagnostic purposes. Such nucleic acids can hybridize with a nucleic acid having a nucleotide sequence specific for β₆ but do not hybridize with nucleic acids encoding non-β₆ proteins, particularly other cell surface receptors. These nucleic acids can readily be determined from the sequence of β₆ and synthesized using a standard nucleic acid synthesizer. Nucleic acids are also provided which specifically hybridize to either the coding or non-coding DNA of β₆.

Integrin cell surface receptors bind ligands, such as extracellular matrix molecules. However, the binding of the integrin to the ligand can be blocked by various means. For example, the binding of a β₆-containing integrin can be blocked by a reagent that binds the β₆ subunit or the β₆-containing integrin. Examples of such reagents include, for example, Arg-Gly-Asp-containing peptides and polypeptides, ligand fragments containing the integrin binding site, as well as antibodies specifically reactive with β₆ or a β₆-containing integrin. Alternatively, the blocking can be carried out by binding the ligand or fragment thereof, recognized by a β₆-containing integrin with a reagent specific for the ligand at a site that inhibits the ligand from binding with the integrin. Since the binding of a β₆-containing integrin to its ligand can mediate cell adhesion to an extracellular matrix molecule, preventing this binding can prevent cell adhesion. Alternatively, cell adhesion can be promoted by increasing the expression of β₆-containing integrins by a cell.

Finally, the invention provides a method of detecting ligands which bind a β₆-containing integrin. The method comprises contacting a β₆-containing integrin with a solution containing ligands suspected of binding β₆-containing integrins. The presence of ligands which bind a β₆-containing integrin is then detected.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE I Identification of a Novel β Subunit

A. Generation of cDNA Fragments by Polymerase Chain Reaction

Tracheal epithelial cells, harvested from male Hartley outbred guinea pigs (Charles River Breeding Laboratories, Bar Harbor, Me.) were grown to confluence over 10-14 days on collagen-impregnated microporous filters commercially available from Costar. RNA was harvested from these primary cultures, and mRNA was purified over oligo(dT)-cellulose columns using the Fast Track AREA isolation kit (Invitrogen, San Diego, Calif.). Two to 5 μg of mRNA was used as a template for cDNA synthesis catalyzed by 200 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.) in a 20-40 μl reaction volume. One to 5 μl of the resultant cDNA was used as a template for polymerase chain reaction (PCR). PCR was carried out in a reaction volume of 25-200 μl. In addition to the template cDNA, each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl (pH 90 at 25° C.), 1.5 mM MgCl₂, 0.01% gelatin, 0.1% Triton X-100, 0.2 mM each of dATP, dGTP, dCTP and dTTP, and 0.05 units/μl Taq DNA polymerase (obtained from either United States Biochemical Corporation, Cleveland, Ohio, or from Promega, Madison, Wis.).

For each reaction, two oligonucleotide primers were also added to obtain final concentration of 1 μM each. The primer pairs are identified below. Each reaction mixture was overlaid with mineral oil, heated to 95° C. for 4 min. in a thermal cycler (Ericomp, San Diego, Calif.), and then subjected to 30 cycles of PCR. Each cycle consisted of 45 seconds at 95° C., 45 seconds at 53° C., and 1 min. at 72° C. Immediately after the last cycle, the sample was maintained at 72° C. for 10 min.

The results of each PCR reaction were analyzed by gel electrophoresis in 1.5% agarose. Reactions that produced fragments of the expected size were electrophoresed in 1.5% low gel temperature agarose (Bio-Rad Laboratories, Richmond, Calif.). The appropriate size band was excised, melted at 68° C., and the DNA was purified by extraction with phenol/chloroform and precipitation in ethanol and ammonium acetate.

B. PCR Primers

To obtain the initial fragment of the novel β subunit cDNA described herein, degenerate mixtures of PCR primers were used. Oligonucleotides were synthesized, trityl-on, by the University of California, San Francisco Biomolecular Resource Center using a DNA synthesizer with standard procedures, and purified over Nen-sorb cartridges (DuPont-New England Nuclear, Boston, Mass.). These consensus primer mixtures were designed to anneal with the nucleotides encoding the highly conserved sequence Asp-Leu-Tyr-Tyr-Leu-Met-Asp-Leu (SEQ ID NO:50) (primer B1F) (SEQ ID NO: 1) and Glu-Gly-Gly-Phe-Asp-Ala-Ile-Met-Gln (SEQ ID NO:53) (primer B2R) (SEQ ID NO:2) that flank an approximately 300-nucleotide region beginning approximately 130 amino acids from the amino terminus of each of the integrin β subunits sequenced to date. The sequences of the primers identified herein are depicted in FIG. 1 (SEQ ID NOS: 1-8).

On the basis of the initial sequence obtained, a specific forward primer was designed to anneal with the sequence encoding the amino acids Pro-Leu-Thr-Asn-Asp-Ala-Glu-Arg (SEQ ID NO:61) (primer BTE2F) (SEQ ID NO:7) ending approximately 49 nucleotides from the 3′ end of the region that had been sequenced. An additional forward primer (B3F) (SEQ ID NO:3) and two reverse primers (B3R and B4R) (SEQ ID NOS:4-5) were also designed to recognize highly conserved consensus regions encoding the sequences Gly-Glu-Cys-Val-Cys-Gly-Gln-Cys (SEQ ID NO:58) (B3 region) (SEQ ID NOS:3-4) and Ile-Gly-Leu-Ala-Leu-Leu-Leu-Ile-Trp-Lys (SEQ ID NO:59) (B4 region) (SEQ ID NO:5). The alignment of these primers with previously published sequences of human β₁, β₂ and β₃ and chicken β₁ is shown in FIG. 1. PCR as described above was performed with cDNA from guinea pig tracheal epithelial cells and the primer pairs BTE2F/B3R (SEQ ID NOS:7 and 4) and B3F/B4R (SEQ ID NOS:3 and 5).

The primer pair BTE2F/B3R (SEQ ID NOS:7 and 4) yielded 1095 additional base pairs of new sequence. Based on this sequence another specific primer (BTE3F) (SEQ ID NO:8) was designed to recognize the sequence Val-Ser-Glu-Asp-Gly-Val (SEQ ID NO:9) near the 3′ end of this sequence, and PCR was performed with this primer in combination with primer B4R (SEQ ID NO:5).

FIG. 1 shows the design of the PCR primers. β subunit consensus primer mixtures were designed on the basis of alignment of published sequences of human β₁, β₂, β₃ and chicken β₁. For forward primers (B1F and B3F) (SEQ ID NOS:1 and 3), the primer sequences included a single nucleotide whenever possible for each of the first two nucleotides of each codon and were usually either degenerate or included deoxyinosine for the third base in codons for amino acids other than methionine. Reverse primers (B2R, B3R, and B4R) (SEQ ID NOS:2, 4 and 5) were designed in the same manner for the complementary DNA strand. Two specific forward primers were designed to recognize β₆ The first (BTE2F) (SEQ ID NO:7) was designed to work across species and was thus degenerate or included deoxyinosine in the third codon position. The second, BTE3F (SEQ ID NO:8), was not degenerate and was designed to only recognize guinea pig β₆.

C. Cloning of Fragments Obtained by PCR

Individual fragments were cloned in pBluescript (Stratagene, San Diego, Calif.) as follows. Purified fragments were resuspended in distilled water containing deoxynucleotides and treated with 2.5 units of DNA polymerase I, large fragment (Promega) to fill in any 3′ recessed ends left after the last cycle of PCR. The 5′ ends were phosphorylated with 5 units of T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.). An aliquot of the above reaction mixture containing approximately 100-200 ng of DNA, was ligated into pBluescript that had been cut with EcoRV (Promega) and dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, Ind.). Ligations were performed at 22° C. for 1 hour with T4 DNA ligase (Bethesda Research Laboratories). The ligation mixture was used to transform competent Escherichia coli (JM109, Clontech, San Francisco, Calif.). Plasmids containing inserts were purified using the Pharmacia miniprep lysis kit (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) denatured in 0.3 M NaOH, further purified over spin columns containing Sephacryl S-400 (Pharmacia), and then sequenced using the Sequenase™ version 2.0 sequencing kit (United States Biochemical Corp., Cleveland, Ohio) and [³⁵S]DATP (Amersham Corp., Arlington Heights, Ill.).

D. Library Screening

PCR fragments generated with the primer pairs B1F/B2R (SEQ ID NOS:1-2) and BTE3F/B4R (SEQ ID NOS:8 and 5) were uniformly labeled with alpha-[³²P]dCTP and used as probes to screen a random-primed cDNA library and an oligo-dT-primed cDNA library. Both libraries were constructed in the plasmid pTZ18R-BstXI obtained from Invitrogen (San Diego, Calif.) from mRNA obtained from the human pancreatic carcinoma cell line FG-2.

Plasmid was purified from clones found to hybridize with either region and inserts were sequenced. A portion of insert DNA from one clone was in turn labeled and used to screen the same libraries. Fourteen independent overlapping clones were sequenced from both ends using primers that recognize regions of the pTZ polylinker. The regions flanking the 3′ end of the putative translated region of the new β subunit were sequenced in both directions from three clones using primers constructed to recognize sequences close to the 3′ end. On the basis of the initial sequences thus obtained, an additional internal sequence was obtained from clones T10, T11, T12 and T14 (FIG. 2) after digestion with specific restriction endonucleases and relegation. Three internal fragments thus generated were subcloned into pBluescript and were also sequenced in both directions. Approximately 90% of the new sequence reported was obtained from both strands of DNA, and 97% was obtained from two or more overlapping clones (FIG. 2).

FIG. 2 shows a map of the sequencing strategy. Shown are the location of clones used to obtain the partial cDNA sequence of guinea pig β₆ (clones 1F, 3L, 3N and 3Y, top) and the complete sequence of human β₆ (clones T1-T19 bottom). Also shown is the location of the translated region (Protein). The location of the transmembrane domain is shown by the letters TM. Clones shown often represent one of several identical clones. Internal sequence of clones with long inserts was obtained by restriction endonuclease digestion and relegation and by ligation of internal fragments into pBluescript. Specific restriction sites employed are shown (Hind, HindIII; Hinc, HincII; Kpn, KpnI; Pst, PstI). The direction and extent of sequencing are shown by arrows. 1109 and 1110 are the sites recognized by oligonucleotide sequencing primers. T18 and T19 each terminated in a poly(A) tail. The regions recognized by the degenerate PCR primers B1F (B1), B2R (B2), B3R/F (B3)., and B4R (B4) and the β₆ primers BTE2F (BTE2) and BTE3F (BTE3) are noted above the guinea pig cDNA map, kb, kilobases.

E. Nucleotide Sequence of a Novel Guinea Pig Integrin β Subunit

PCR using cDNA from guinea pig airway epithelial cells and the consensus primer mixtures B1F and B2R (FIG. 1) amplified DNA fragments with the expected size of approximately 350 nucleotides. When the fragment DNA was sequenced after cloning into pBluescript, recombinant clones each contained inserts with one of two distinct sequences. One sequence encoded a stretch of 98 amino acids that was 97% identical to the expected region of human β₁ and was therefore presumed to be guinea pig β₁. The other sequence encoded 98 amino acids that were only 53% identical to human β₁, 45% identical to human β₂, and 57% identical to human β₃ (FIG. 2, clone 1F). Both of the guinea pig sequences included the integrin β subunit consensus sequences Ser-X-Ser-Met-X-Asp-Asp-Leu (SEQ ID NO:46) and Gly-Phe-Gly-Ser-Phe-Val (SEQ ID NO:47), and both contained the 2 cysteine residues found in this region in all known integrin β subunits. These data suggest that one of the two sequences we obtained encoded a new member of the integrin β subunit family.

This novel sequence was extended by further PCR steps utilizing primers specific for the novel sequence (BTE2F, BTE3F) (SEQ ID NOS:7 and 8) in combination with two additional degenerate primers (B3R and B4R, see FIGS. 1, 2 and 4). With the primer pair BTE2F/B3R (SEQ ID NOS:7 and 4) two different cDNA products were obtained (3L and 3N in FIG. 2) due to an unexpected hybridization of the B3R primer with a site 220 nucleotides further downstream (B3′ in FIG. 2). The 1732-nucleotide sequence determined from these clones is shown in FIG. 3.

FIG. 3 shows the nucleotide sequences and corresponding amino acid sequences for human (H) β₆ (SEQ ID NOS:26-27) and guinea pig (GP) β₆ (SEQ ID NOS:28-29). The amino acid translation is denoted by the single letter code beneath the second nucleotide of each codon from the translated region of human β₆. For the guinea pig sequence, only amino acids that differ from the human sequence are shown. The numbers along the right-hand margin denote the nucleotide or amino acid number of the last entry on each line. The numbering system used starts with the first nucleotide or amino acid available for each sequence shown. The nine potential sites for N-glycosylation in the putative extracellular domain of human β₆ are underlined.

F. Nucleotide Sequence of Human β₆

Screening of cDNA libraries constructed from the human pancreatic carcinoma cell line FG-2 with guinea pig cDNA probes 1F and 3Y (see FIG. 2) and subsequent screening with a probe constructed from a portion of clone T10 (FIG. 2) produced 14 independent positive clones. The two longest clones (T18 and T19) extended to the poly(A) tail. A map of these clones, constructed on the basis of sequence information and of the mobility of inserts cut out of these clones in agarose gels is shown in FIG. 2. This map predicts an mRNA of approximately 5 kilobases including at least a 226-nucleotide untranslated region at the 5′ end and, a 2364-nucleotide open reading frame, and a 3′ untranslated region of approximately 2.5 kilobases. This molecule has been termed integrin β₆.

FIG. 3 shows the partial nucleotide and complete amino acid sequences for human β₆ (SEQ ID NOS:26-27) (excluding most of the 3′-untranslated region) and the alignment of the 1732 nucleotides of sequence obtained from PCR of guinea pig airway epithelial cell cDNA. Of the 577 amino acids deduced from the region sequenced in both species only 36 residues differ; the amino acid sequences are 94% identical. Furthermore, of the 1732 nucleotides sequenced in both species, 91% are identical. Nine potential glycosylation sites present in the putative extracellular domain of human β₆ are shown by underlining. All seven of these sites that lie within the 577 amino acids obtained for guinea pig β₆ are also present in the guinea pig protein. If all of the potential glycosylation sites are occupied with oligosaccharides having an average molecular weight of 2,500, the predicted molecular weight of human β₆ would be 106,000.

Comparison of the 788-amino acid sequence deduced from the open reading frame to the three previously sequenced human β subunits (SEQ ID NOS:30-32) and the myospheroid protein of Drosophila (SEQ ID NO:33) is shown in FIG. 4.

FIG. 4 shows the alignment of β₆ with four previously reported integrin β subunits. Previously published sequences for human β₁ (SEQ ID NO:30), human β₂ (SEQ ID NO:31), human β₃ (SEQ ID NO:32), the myospheroid gene product (βmyo) of Drosophila (SEQ ID NO:33), and the novel sequence described as β₆ (SEQ ID NO:27) are shown using the single letter amino acid code. The 56 conserved cysteines are noted by * and the 120 other invariant amino acids by=above each line. The transmembrane domain is underlined. The regions used for constructing the consensus subunit primers B1F (B1) (SEQ ID NO:1), B2R (B2) (SEQ ID NO:2), B3F/R (B3) (SEQ ID NOS:3-4), and B4R (B4) (SEQ ID NO:5) are labeled below the alignment in bold type. The numbers along the right-hand margin denote the number of the last amino acid in each line beginning from the first amino acid of each putative signal sequence.

There are 179 amino acid residues that are identical in each of the other β subunits and in β₆ including 56 conserved cysteine residues. The overall percentage of identical amino acids between β₆ and the other human β subunits is 47% for β₃, 42% for β₁ and 38% for β₂. Human β₆ is also 39% identical to the Drosophila β subunit. Human β₁, β₂ and β₃ and the Drosophila β subunit all have cytoplasmic regions consisting of 41 amino acids (beginning after the putative transmembrane domain shown by the underline in FIG. 4). Although β₆ contains each of the 10 conserved amino acid residues in this cytoplasmic region it also contains an 11-amino acid extension at the carboxyl terminus. β₆ also contains two Arg-Gly-Asp sequences, one at amino acids 514-516 and the other at 594-596. These regions could serve as recognition sites for other ligands of the integrin family.

PCR using the primer pair B3F/B4R (SEQ ID NOS:3 and 5) (see FIG. 1) amplified fragments of the expected size of approximately 750 nucleotides. Cloning and sequencing of the fragments did not result in any additional clones containing the novel β subunit sequence but did result in several clones with inserts encoding an amino acid sequence that was 97% identical to the corresponding region of human β₃ and several others encoding an amino acid sequence that was 93% identical to human β₁ (SEQ ID NO:35) (FIG. 5). These are presumably the guinea pig homologues of β₁ (SEQ ID NO:37) and β₃(SEQ ID NO:41), respectively. The nucleotide sequences of guinea pig (SEQ ID NO:36) and human β (SEQ ID NO:34) are 80% identical, and those of guinea pig (SEQ ID NO:40) and human β₃ (SEQ ID NO:38) are 91% identical.

FIG. 5 shows the alignment of partial nucleotide and amino acid sequences from human (H) and guinea pig (GP) β₁ (SEQ ID NOS:34-37), β₃ (SEQ ID NOS:38-41), and β₆ (SEQ ID NOS:42-45) for the region just downstream from the B3F primer. Amino acid translations denoted by the one-letter code are shown below the second nucleotide of each codon. For the guinea pig sequences, only amino acids that differ from the human sequences are shown. The numbers shown along the right-hand margin denote the nucleotide number for human β₆. The sequences for human β₁ and β₃ are from previously published reports.

EXAMPLE II β₆ Associates with α_(V) and α_(F) Subunits

To determine that the novel β subunit of the present invention is associated with an α chain similar to other known integrins, antisera against peptides from the cytoplasmic domain sequence of β₆ were prepared. The following amino acid peptides from the cytoplasmic sequence of β₆ were prepared and used to immunize rabbits: RGSTSTFKNVTYKHR (SEQ ID NO:48) (residues 763-777) and YKHREKQKVDLSTDC (SEQ ID NO:49 (residues 774-788). The antisera were raised in rabbits according to standard procedures known in the art. Briefly, peptides were chemically coupled to keyhole lympet hemocyanin, and were injected in rabbits in either complete (first injection only) or incomplete Freund's adjuvant as described, for example, in Antibodies: A Laboratory Manual, E. Harlow and D. Lowe, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 11724. Antisera were termed 6830 (to peptides corresponding to residues 763-777) and 6341 (to peptides corresponding to residues 774-788).

The resulting polyclonal antibodies were used to immunoprecipitate detergent lysates from the pancreatic carcinoma cell line FG-2 that had been surface radioiodinated according to procedures well known in the art such as described, for example, in Kajiji et al., EMBO J. 3:673-680 (1989). A complex of two bands was precipitated of respectively 150 kilodaltons (Kd) and 97 Kd in SDS-PAGE under non-reducing conditions. Under reducing conditions, the two bands migrated as a diffused band, extending from 130 Kd to 116 Kd. These bands were specific since pre-immune serum did not precipitate any of them and they were not present when the immunoprecipitation was carried out in the presence of the corresponding immunogenic peptide. Furthermore, the same complex of two bands was precipitated by both the 6830 and 6841 antibodies, which were raised against independent peptides from the cytoplasmic sequence deduced from β₆ cDNA clones.

To determine which of the two precipitated bands corresponds to β₆, a SDS-heat denaturated lysate from surface-radioiodinated FG-2 cells was immunoprecipitated with the 6841 antibody. Only the 97 Kd band was detectable (non-reducing conditions), identifying it as the β₆ band. Under reducing conditions, the apparent molecular weight of this band increased to 116 Kd suggesting the presence of many intra-chain disulfide bonds, which is consistent with the primary structure of β₆ and of other integrin β chains.

The other band, of 150 Kd or 130 Kd under non-reducing or reducing conditions, respectively, is likely to be an α subunit since it dissociates after SDS-heat denaturation of the lysate, indicating that it is non-covalently associated with the β₆ polypeptide. Furthermore, similar to certain other integrin a chains, its molecular weight decreases under reducing conditions by about 20 Kd (130 Kd versus 150 Kd under non-reducing conditions) probably due to a disulfide linked small peptide that dissociates upon reduction.

To identify which a chain is associated with β₆, the αβ₆ integrin complex was purified by immuno-affinity chromatography on a 6841-protein A sepharose matrix according to procedures welL known in the art such as described, for example, in Kajiji et al., EMBO J. 3:673-680 (1989). The eluted material was immunoprecipitated with antibodies specific for α₁, α₂, α₃, α₅, α₆ and α_(V) which are known to be expressed in FG-2 cells. Only the anti-α_(V) monoclodinal antibody 142.19, obtained from Dr. David Cheresh, The Scripps Research Institution, La Jolla, Calif., reacted with the purified material, which indicates that the α_(V) is associated with β₆ in this pancreatic carcinoma cell line.

To confirm this data, immunodepletion experiments on surface-radioiodinated FG-2 lysates were performed according to methods well known in the art such as described in Kajiji et al., EMBO J. 3:673-680 (1989). The cell lysate was depleted with the 6841 anti-β₆ antibody or, in parallel, with a control antiserum, and then immunoprecipitated with the 142.19 anti-α_(V) antibody. A smaller amount of α_(V) was present in the immunoprecipitation on the β₆ depleted lysate and no 97 Kd β₆ band was visible. Instead, a smaller band of about 90 Kd was present. It is hypothesized that this smaller band represents the β₆ chain also associated with α_(V) in these cells. In the control lysate depleted with normal rabbit serum, all three bands, 150 Kd (α_(V)), 97 Kd (β₆) and 90 Kd (β₅) were present after immunoprecipitation with the anti-α_(V) 142.19 antibody.

Another immunodepletion was carried out using 142.19 antibody as the depleting antibody, or in parallel a mouse monoclonal as a control antibody. Immunoprecipitations of α_(V)-depleted lysate with anti-α_(V) 142.19 antibodies did not show the presence of any band, indicating that all α_(V)-containing integrins had been removed. However, the 6841 anti-β₆ antibody still precipitated a complex of two bands, one corresponding to β₆ the other with a molecular weight close to that of α₆ This α chain, however, must differ from α_(V) since it is unreactive with anti-α_(V) monoclonal antibodies and is referred to herein as α_(F). In the control depleted lysates, the 6841 anti-β₆ antibody precipitates much stronger bands, consistent with the possibility that, in FG-2 cells, two β₆ integrins exist, α_(V)β₆ and α_(F)β₆.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

43 23 base pairs nucleic acid single linear 1 GACCTCTACT ACCTGATGGA CCT 23 25 base pairs nucleic acid single linear misc_difference replace(5, “”) /standard_name= “N=INOSINE” misc_difference replace(14, “”) /standard_name= “N=INOSINE” misc_difference replace(17, “”) /standard_name= “N=INOSINE” 2 GCATNATGGC ATCNAANCCA CCTTC 25 23 base pairs nucleic acid single linear misc_difference replace(3, “”) /standard_name= “N=INOSINE” misc_difference replace(6, “”) /standard_name= “N=INOSINE” misc_difference replace(10, “”) /standard_name= “N=INOSINE” misc_difference replace(18, “”) /standard_name= “N=INOSINE” 3 GGNGANTGTN TTTGTGGNCA GTG 23 20 base pairs nucleic acid single linear misc_difference replace(6, “”) /standard_name= “N=INOSINE” misc_difference replace(14, “”) /standard_name= “N=INOSINE” misc_difference replace(18, “”) /standard_name= “N=INOSINE” 4 CACTGNCCAC AAANACANTC 20 29 base pairs nucleic acid single linear misc_difference replace(6, “”) /standard_name= “N=INOSINE” misc_difference replace(11, “”) /standard_name= “N=INOSINE” misc_difference replace(15, “”) /standard_name= “N=INOSINE” misc_difference replace(18, “”) /standard_name= “N=INOSINE” misc_difference replace(24, “”) /standard_name= “N=INOSINE” 5 TTCCANATCA NTAGNGCNAC AAGNCCAAT 29 24 base pairs nucleic acid single linear 6 CCATTGACAA ATGATGCTGA AAGA 24 24 base pairs nucleic acid single linear misc_difference replace(3, “”) /standard_name= “N=INOSINE” misc_difference replace(6, “”) /standard_name= “N=INOSINE” misc_difference replace(9, “”) /standard_name= “N=INOSINE” misc_difference replace(18, “”) /standard_name= “N=INOSINE” 7 CCNTTNACNA ATGATGCNGA AAGA 24 17 base pairs nucleic acid single linear 8 CATCTCCGAA GACGGCA 17 17 base pairs nucleic acid single linear 9 CATCTCCGAA GACGGCA 17 23 base pairs nucleic acid single linear 10 GACCTGTACT ATCTGATGGA CCT 23 23 base pairs nucleic acid single linear 11 GACATCTACT ACTTGATGGA CCT 23 23 base pairs nucleic acid single linear 12 GACCTCTACT ACCTTATGGA CCT 23 23 base pairs nucleic acid single linear 13 GACCTTTATT ATCTTATGGA CCT 23 26 base pairs nucleic acid single linear 14 GAGGGTGGGC TGGACGCCAT GATGCA 26 26 base pairs nucleic acid single linear 15 GAGGGTGGCT TTGATGCCAT CATGCA 26 26 base pairs nucleic acid single linear 16 GAAGGTGGTT TCGATGCCAT CATGCA 26 26 base pairs nucleic acid single linear 17 GAAGGTGGAT TTGATGCAAT AATGCA 26 24 base pairs nucleic acid single linear 18 GGGGACTGTG TCTGCGGGCA GTGC 24 24 base pairs nucleic acid single linear 19 GGCGAGTGCC TCTGTGGTCA ATGT 24 24 base pairs nucleic acid single linear 20 GGAGAGTGCG TCTGCGGACA GTGT 24 24 base pairs nucleic acid single linear 21 GGAGAGTGCA TTTGCGGACA GTGC 24 30 base pairs nucleic acid single linear 22 ATCGGCATTC TCCTGCTGGT CATCTGGAAG 30 30 base pairs nucleic acid single linear 23 ATTGGCCTTG CCGCCCTGCT CATCTGGAAA 30 30 base pairs nucleic acid single linear 24 ATTGGCCTTG CATTACTGCT GATATGGAAG 30 30 base pairs nucleic acid single linear 25 ATTGGACTTG CATTGTTATT GATTTGGAAA 30 2644 base pairs nucleic acid double linear CDS 227..2593 26 TAAACACAGC TTTTCTGCTT TACCTGTCCA GGTAGCCTCT GTTTTCATTT CAGTCTTAAT 60 GAAAACTTTC TAACTTATAT CTCAAGTTTC TTTTCAAAGC AGTGTAAGTA GTATTTAAAA 120 TGTTATACTT CAAGAAAGAA AGACTTTAAC GATATTCAGC GTTGGTCTTG TAACGCTGAA 180 GGTAATTCAT TTTTTAATCG GTCTCGCACA GCAAGAACTG AAACGA ATG GGG ATT 235 Met Gly Ile 1 GAA CTG CTT TGC CTG TTC TTT CTA TTT CTA GGA AGG AAT GAT TCA CGT 283 Glu Leu Leu Cys Leu Phe Phe Leu Phe Leu Gly Arg Asn Asp Ser Arg 5 10 15 ACA AGG TGG CTG TGC CTG GGA GGT GCA GAA ACC TGT GAA GAC TGC CTG 331 Thr Arg Trp Leu Cys Leu Gly Gly Ala Glu Thr Cys Glu Asp Cys Leu 20 25 30 35 CTT ATT GGA CCT CAG TGT GCC TGG TGT GCT CAG GAG AAT TTT ACT CAT 379 Leu Ile Gly Pro Gln Cys Ala Trp Cys Ala Gln Glu Asn Phe Thr His 40 45 50 CCA TCT GGA GTT GGC GAA AGG TGT GAT ACC CCA GCA AAC CTT TTA GCT 427 Pro Ser Gly Val Gly Glu Arg Cys Asp Thr Pro Ala Asn Leu Leu Ala 55 60 65 AAA GGA TGT CAA TTA AAC TTC ATC GAA AAC CCT GTC TCC CAA GTA GAA 475 Lys Gly Cys Gln Leu Asn Phe Ile Glu Asn Pro Val Ser Gln Val Glu 70 75 80 ATA CTT AAA AAT AAG CCT CTC AGT GTA GGC AGA CAG AAA AAT AGT TCT 523 Ile Leu Lys Asn Lys Pro Leu Ser Val Gly Arg Gln Lys Asn Ser Ser 85 90 95 GAC ATT GTT CAG ATT GCA CCT CAA AGC TTG ATC CTT AAG TTG AGA CCA 571 Asp Ile Val Gln Ile Ala Pro Gln Ser Leu Ile Leu Lys Leu Arg Pro 100 105 110 115 GGT GGT GCG CAG ACT CTG CAG GTG CAT GTC CGC CAG ACT GAG GAC TAC 619 Gly Gly Ala Gln Thr Leu Gln Val His Val Arg Gln Thr Glu Asp Tyr 120 125 130 CCG GTG GAT TTG TAT TAC CTC ATG GAC CTC TCC GCC TCC ATG GAT GAC 667 Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser Met Asp Asp 135 140 145 GAC CTC AAC ACA ATA AAG GAG CTG GGC TCC GGC CTT TCC AAA GAG ATG 715 Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser Gly Leu Ser Lys Glu Met 150 155 160 TCT AAA TTA ACC AGC AAC TTT AGA CTG GGC TTC GGA TCT TTT GTG GAA 763 Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser Phe Val Glu 165 170 175 AAA CCT GTA TCC CCT TTT GTG AAA ACA ACA CCA GAA GAA ATT GCC AAC 811 Lys Pro Val Ser Pro Phe Val Lys Thr Thr Pro Glu Glu Ile Ala Asn 180 185 190 195 CCT TGC AGT AGT ATT CCA TAC TTC TGT TTA CCT ACA TTT GGA TTC AAG 859 Pro Cys Ser Ser Ile Pro Tyr Phe Cys Leu Pro Thr Phe Gly Phe Lys 200 205 210 CAC ATT TTG CCA TTG ACA AAT GAT GCT GAA AGA TTC AAT GAA ATT GTG 907 His Ile Leu Pro Leu Thr Asn Asp Ala Glu Arg Phe Asn Glu Ile Val 215 220 225 AAG AAT CAG AAA ATT TCT GCT AAT ATT GAC ACA CCC GAA GGT GGA TTT 955 Lys Asn Gln Lys Ile Ser Ala Asn Ile Asp Thr Pro Glu Gly Gly Phe 230 235 240 GAT GCA ATT ATG CAA GCT GCT GTG TGT AAG GAA AAA ATT GGC TGG CGG 1003 Asp Ala Ile Met Gln Ala Ala Val Cys Lys Glu Lys Ile Gly Trp Arg 245 250 255 AAT GAC TCC CTC CAC CTC CTG GTC TTT GTG AGT GAT GCT GAT TCT CAT 1051 Asn Asp Ser Leu His Leu Leu Val Phe Val Ser Asp Ala Asp Ser His 260 265 270 275 TTT GGA ATG GAC AGC AAA CTA GCA GGC ATC GTC ATT CCT AAT GAC GGG 1099 Phe Gly Met Asp Ser Lys Leu Ala Gly Ile Val Ile Pro Asn Asp Gly 280 285 290 CTC TGT CAC TTG GAC AGC AAG AAT GAA TAC TCC ATG TCA ACT GTC TTG 1147 Leu Cys His Leu Asp Ser Lys Asn Glu Tyr Ser Met Ser Thr Val Leu 295 300 305 GAA TAT CCA ACA ATT GGA CAA CTC ATT GAT AAA CTG GTA CAA AAC AAC 1195 Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp Lys Leu Val Gln Asn Asn 310 315 320 GTG TTA TTG ATC TTC GCT GTA ACC CAA GAA CAA GTT CAT TTA TAT GAG 1243 Val Leu Leu Ile Phe Ala Val Thr Gln Glu Gln Val His Leu Tyr Glu 325 330 335 AAT TAC GCA AAA CTT ATT CCT GGA GCT ACA GTA GGT CTA CTT CAG AAG 1291 Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr Val Gly Leu Leu Gln Lys 340 345 350 355 GAC TCC GGA AAC ATT CTC CAG CTG ATC ATC TCA GCT TAT GAA GAA CTG 1339 Asp Ser Gly Asn Ile Leu Gln Leu Ile Ile Ser Ala Tyr Glu Glu Leu 360 365 370 CGG TCT GAG GTG GAA CTG GAA GTA TTA GGA GAC ACT GAA GGA CTC AAC 1387 Arg Ser Glu Val Glu Leu Glu Val Leu Gly Asp Thr Glu Gly Leu Asn 375 380 385 TTG TCA TTT ACA GCC ATC TGT AAC AAC GGT ACC CTC TTC CAA CAC CAA 1435 Leu Ser Phe Thr Ala Ile Cys Asn Asn Gly Thr Leu Phe Gln His Gln 390 395 400 AAG AAA TGC TCT CAC ATG AAA GTG GGA GAC ACA GCT TCC TTC AGC GTG 1483 Lys Lys Cys Ser His Met Lys Val Gly Asp Thr Ala Ser Phe Ser Val 405 410 415 ACT GTG AAT ATC CCA CAC TGC GAG AGA AGA AGC AGG CAC ATT ATC ATA 1531 Thr Val Asn Ile Pro His Cys Glu Arg Arg Ser Arg His Ile Ile Ile 420 425 430 435 AAG CCT GTG GGG CTG GGG GAT GCC CTG GAA TTA CTT GTC AGC CCA GAA 1579 Lys Pro Val Gly Leu Gly Asp Ala Leu Glu Leu Leu Val Ser Pro Glu 440 445 450 TGC AAC TGC GAC TGT CAG AAA GAA GTG GAA GTG AAC AGC TCC AAA TGT 1627 Cys Asn Cys Asp Cys Gln Lys Glu Val Glu Val Asn Ser Ser Lys Cys 455 460 465 CAC CAC GGG AAC GGC TCT TTC CAG TGT GGG GTG TGT GCC TGC CAC CCT 1675 His His Gly Asn Gly Ser Phe Gln Cys Gly Val Cys Ala Cys His Pro 470 475 480 GGC CAC ATG GGG CCT CGC TGT GAG TGT GGC GAG GAC ATG CTG AGC ACA 1723 Gly His Met Gly Pro Arg Cys Glu Cys Gly Glu Asp Met Leu Ser Thr 485 490 495 GAT TCC TGC AAG GAG GCC CCA GAT CAT CCC TCC TGC AGC GGA AGG GGT 1771 Asp Ser Cys Lys Glu Ala Pro Asp His Pro Ser Cys Ser Gly Arg Gly 500 505 510 515 GAC TGC TAC TGT GGG CAG TGT ATC TGC CAC TTG TCT CCC TAT GGA AAC 1819 Asp Cys Tyr Cys Gly Gln Cys Ile Cys His Leu Ser Pro Tyr Gly Asn 520 525 530 ATT TAT GGA CCT TAT TGC CAG TGT GAC AAT TTC TCC TGC GTG AGA CAC 1867 Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn Phe Ser Cys Val Arg His 535 540 545 AAA GGG CTG CTC TGC GGA GGT AAC GGC GAC TGT GAC TGT GGT GAA TGT 1915 Lys Gly Leu Leu Cys Gly Gly Asn Gly Asp Cys Asp Cys Gly Glu Cys 550 555 560 GTG TGC AGG AGC GGC TGG ACT GGC GAG TAC TGC AAC TGC ACC ACC AGC 1963 Val Cys Arg Ser Gly Trp Thr Gly Glu Tyr Cys Asn Cys Thr Thr Ser 565 570 575 ACG GAC TCC TGC GTC TCT GAA GAT GGA GTG CTC TGC AGC GGG CGC GGG 2011 Thr Asp Ser Cys Val Ser Glu Asp Gly Val Leu Cys Ser Gly Arg Gly 580 585 590 595 GAC TGT GTT TGT GGC AAG TGT GTT TGC ACA AAC CCT GGA GCC TCA GGA 2059 Asp Cys Val Cys Gly Lys Cys Val Cys Thr Asn Pro Gly Ala Ser Gly 600 605 610 CCA ACC TGT GAA CGA TGT CCT ACC TGT GGT GAC CCC TGT AAC TCT AAA 2107 Pro Thr Cys Glu Arg Cys Pro Thr Cys Gly Asp Pro Cys Asn Ser Lys 615 620 625 CGG AGC TGC ATT GAG TGC CAC CTG TCA GCA GCT GGC CAA GCC GGA GAA 2155 Arg Ser Cys Ile Glu Cys His Leu Ser Ala Ala Gly Gln Ala Gly Glu 630 635 640 GAA TGT GTG GAC AAG TGC AAA CTA GCT GGT GCG ACC ATC AGT GAA GAA 2203 Glu Cys Val Asp Lys Cys Lys Leu Ala Gly Ala Thr Ile Ser Glu Glu 645 650 655 GAA GAT TTC TCA AAG GAT GGT TCT GTT TCC TGC TCT CTG CAA GGA GAA 2251 Glu Asp Phe Ser Lys Asp Gly Ser Val Ser Cys Ser Leu Gln Gly Glu 660 665 670 675 AAT GAA TGT TTA ATT ACA TTC CTA ATA ACT ACA GAT AAT GAG GGG AAA 2299 Asn Glu Cys Leu Ile Thr Phe Leu Ile Thr Thr Asp Asn Glu Gly Lys 680 685 690 ACC ATC ATT CAC AGC ATC AAT GAA AAA GAT TGT CCG AAG CCT CCA AAC 2347 Thr Ile Ile His Ser Ile Asn Glu Lys Asp Cys Pro Lys Pro Pro Asn 695 700 705 ATT CCC ATG ATC ATG TTA GGG GTT TCC CTG GCT ACT CTT CTC ATC GGG 2395 Ile Pro Met Ile Met Leu Gly Val Ser Leu Ala Thr Leu Leu Ile Gly 710 715 720 GTT GTC CTA CTG TGC ATC TGG AAG CTA CTG GTG TCA TTT CAT GAT CGT 2443 Val Val Leu Leu Cys Ile Trp Lys Leu Leu Val Ser Phe His Asp Arg 725 730 735 AAA GAA GTT GCC AAA TTT GAA GCA GAA CGA TCA AAA GCC AAG TGG CAA 2491 Lys Glu Val Ala Lys Phe Glu Ala Glu Arg Ser Lys Ala Lys Trp Gln 740 745 750 755 ACG GGA ACC AAT CCA CTC TAC AGA GGA TCC ACA AGT ACT TTT AAA AAT 2539 Thr Gly Thr Asn Pro Leu Tyr Arg Gly Ser Thr Ser Thr Phe Lys Asn 760 765 770 GTA ACT TAT AAA CAC AGG GAA AAA CAA AAG GTA GAC CTT TCC ACA GAT 2587 Val Thr Tyr Lys His Arg Glu Lys Gln Lys Val Asp Leu Ser Thr Asp 775 780 785 TGC TAGAACTACT TTATGCATAA AAAAAGTCTG TTTCACTGAT ATGAAATGTT AATG 2644 Cys 788 amino acids amino acid linear protein 27 Met Gly Ile Glu Leu Leu Cys Leu Phe Phe Leu Phe Leu Gly Arg Asn 1 5 10 15 Asp Ser Arg Thr Arg Trp Leu Cys Leu Gly Gly Ala Glu Thr Cys Glu 20 25 30 Asp Cys Leu Leu Ile Gly Pro Gln Cys Ala Trp Cys Ala Gln Glu Asn 35 40 45 Phe Thr His Pro Ser Gly Val Gly Glu Arg Cys Asp Thr Pro Ala Asn 50 55 60 Leu Leu Ala Lys Gly Cys Gln Leu Asn Phe Ile Glu Asn Pro Val Ser 65 70 75 80 Gln Val Glu Ile Leu Lys Asn Lys Pro Leu Ser Val Gly Arg Gln Lys 85 90 95 Asn Ser Ser Asp Ile Val Gln Ile Ala Pro Gln Ser Leu Ile Leu Lys 100 105 110 Leu Arg Pro Gly Gly Ala Gln Thr Leu Gln Val His Val Arg Gln Thr 115 120 125 Glu Asp Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu Ser Ala Ser 130 135 140 Met Asp Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser Gly Leu Ser 145 150 155 160 Lys Glu Met Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly Phe Gly Ser 165 170 175 Phe Val Glu Lys Pro Val Ser Pro Phe Val Lys Thr Thr Pro Glu Glu 180 185 190 Ile Ala Asn Pro Cys Ser Ser Ile Pro Tyr Phe Cys Leu Pro Thr Phe 195 200 205 Gly Phe Lys His Ile Leu Pro Leu Thr Asn Asp Ala Glu Arg Phe Asn 210 215 220 Glu Ile Val Lys Asn Gln Lys Ile Ser Ala Asn Ile Asp Thr Pro Glu 225 230 235 240 Gly Gly Phe Asp Ala Ile Met Gln Ala Ala Val Cys Lys Glu Lys Ile 245 250 255 Gly Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe Val Ser Asp Ala 260 265 270 Asp Ser His Phe Gly Met Asp Ser Lys Leu Ala Gly Ile Val Ile Pro 275 280 285 Asn Asp Gly Leu Cys His Leu Asp Ser Lys Asn Glu Tyr Ser Met Ser 290 295 300 Thr Val Leu Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp Lys Leu Val 305 310 315 320 Gln Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln Glu Gln Val His 325 330 335 Leu Tyr Glu Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr Val Gly Leu 340 345 350 Leu Gln Lys Asp Ser Gly Asn Ile Leu Gln Leu Ile Ile Ser Ala Tyr 355 360 365 Glu Glu Leu Arg Ser Glu Val Glu Leu Glu Val Leu Gly Asp Thr Glu 370 375 380 Gly Leu Asn Leu Ser Phe Thr Ala Ile Cys Asn Asn Gly Thr Leu Phe 385 390 395 400 Gln His Gln Lys Lys Cys Ser His Met Lys Val Gly Asp Thr Ala Ser 405 410 415 Phe Ser Val Thr Val Asn Ile Pro His Cys Glu Arg Arg Ser Arg His 420 425 430 Ile Ile Ile Lys Pro Val Gly Leu Gly Asp Ala Leu Glu Leu Leu Val 435 440 445 Ser Pro Glu Cys Asn Cys Asp Cys Gln Lys Glu Val Glu Val Asn Ser 450 455 460 Ser Lys Cys His His Gly Asn Gly Ser Phe Gln Cys Gly Val Cys Ala 465 470 475 480 Cys His Pro Gly His Met Gly Pro Arg Cys Glu Cys Gly Glu Asp Met 485 490 495 Leu Ser Thr Asp Ser Cys Lys Glu Ala Pro Asp His Pro Ser Cys Ser 500 505 510 Gly Arg Gly Asp Cys Tyr Cys Gly Gln Cys Ile Cys His Leu Ser Pro 515 520 525 Tyr Gly Asn Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn Phe Ser Cys 530 535 540 Val Arg His Lys Gly Leu Leu Cys Gly Gly Asn Gly Asp Cys Asp Cys 545 550 555 560 Gly Glu Cys Val Cys Arg Ser Gly Trp Thr Gly Glu Tyr Cys Asn Cys 565 570 575 Thr Thr Ser Thr Asp Ser Cys Val Ser Glu Asp Gly Val Leu Cys Ser 580 585 590 Gly Arg Gly Asp Cys Val Cys Gly Lys Cys Val Cys Thr Asn Pro Gly 595 600 605 Ala Ser Gly Pro Thr Cys Glu Arg Cys Pro Thr Cys Gly Asp Pro Cys 610 615 620 Asn Ser Lys Arg Ser Cys Ile Glu Cys His Leu Ser Ala Ala Gly Gln 625 630 635 640 Ala Gly Glu Glu Cys Val Asp Lys Cys Lys Leu Ala Gly Ala Thr Ile 645 650 655 Ser Glu Glu Glu Asp Phe Ser Lys Asp Gly Ser Val Ser Cys Ser Leu 660 665 670 Gln Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu Ile Thr Thr Asp Asn 675 680 685 Glu Gly Lys Thr Ile Ile His Ser Ile Asn Glu Lys Asp Cys Pro Lys 690 695 700 Pro Pro Asn Ile Pro Met Ile Met Leu Gly Val Ser Leu Ala Thr Leu 705 710 715 720 Leu Ile Gly Val Val Leu Leu Cys Ile Trp Lys Leu Leu Val Ser Phe 725 730 735 His Asp Arg Lys Glu Val Ala Lys Phe Glu Ala Glu Arg Ser Lys Ala 740 745 750 Lys Trp Gln Thr Gly Thr Asn Pro Leu Tyr Arg Gly Ser Thr Ser Thr 755 760 765 Phe Lys Asn Val Thr Tyr Lys His Arg Glu Lys Gln Lys Val Asp Leu 770 775 780 Ser Thr Asp Cys 785 1732 base pairs nucleic acid double linear CDS 1..1731 28 TCC GCC TCC ATG GAC GAT GAC CTC AAC ACA ATC AAA GAG CTG GGC TCC 48 Ser Ala Ser Met Asp Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser 1 5 10 15 CTG CTT TCA AAG GAG ATG TCT AAA TTA ACT AGC AAC TTT AGA CTG GGC 96 Leu Leu Ser Lys Glu Met Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly 20 25 30 TTC GGC TCT TTT GTA GAA AAA CCC GTC TCC CCT TTT ATG AAA ACA ACA 144 Phe Gly Ser Phe Val Glu Lys Pro Val Ser Pro Phe Met Lys Thr Thr 35 40 45 CCA GAG GAA ATT GCC AAC CCT TGC AGT AGT ATT CCA TAT ATC TGC TTA 192 Pro Glu Glu Ile Ala Asn Pro Cys Ser Ser Ile Pro Tyr Ile Cys Leu 50 55 60 CCT ACA TTT GGA TTC AAG CAC ATT CTG CCA TTG ACA AAT GAT GCT GAA 240 Pro Thr Phe Gly Phe Lys His Ile Leu Pro Leu Thr Asn Asp Ala Glu 65 70 75 80 AGA TTC AAT GAA ATT GTG AAG AAA CAG AAA ATT TCT GCT AAT ATT GAC 288 Arg Phe Asn Glu Ile Val Lys Lys Gln Lys Ile Ser Ala Asn Ile Asp 85 90 95 AAC CCT GAA GGT GGA TTC GAC GCC ATT ATG CAA GCT GCT GTG TGT AAG 336 Asn Pro Glu Gly Gly Phe Asp Ala Ile Met Gln Ala Ala Val Cys Lys 100 105 110 GAA AAA ATT GGC TGG CGG AAT GAT TCG CTC CAT CTC CTA GTC TTC GTG 384 Glu Lys Ile Gly Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe Val 115 120 125 AGT GAT GCC GAT TCT CAT TTT GGA ATG GAC AGC AAA CTG GCA GGC ATT 432 Ser Asp Ala Asp Ser His Phe Gly Met Asp Ser Lys Leu Ala Gly Ile 130 135 140 GTC ATT CCC AAC GAT GGG CTG TGT CAC TTG GAC AGC AAG AAT GAA TAC 480 Val Ile Pro Asn Asp Gly Leu Cys His Leu Asp Ser Lys Asn Glu Tyr 145 150 155 160 TCC ATG TCA ACT GTC ATG GAA TAT CCA ACA ATT GGA CAA CTC ATT GAT 528 Ser Met Ser Thr Val Met Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp 165 170 175 AAA GTG GTA CAA AAC AAT GTG TTA CTG ATC TTT GCT GTA ACC CAA GAA 576 Lys Val Val Gln Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln Glu 180 185 190 CAA GTT CCA CTA TAT GAG AAT TAT GCA AAA CTT ATT CCT GGA GCC ACA 624 Gln Val Pro Leu Tyr Glu Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr 195 200 205 GTG GGG CTA CTT CAC AAG GAC TCT GGA AAC ATT CTC CAA CTG ATC ATC 672 Val Gly Leu Leu His Lys Asp Ser Gly Asn Ile Leu Gln Leu Ile Ile 210 215 220 TCA GCT TAT GAA GAA CTG CGG TCT GAG GTG GAG CTG GAA GTA TTA GGA 720 Ser Ala Tyr Glu Glu Leu Arg Ser Glu Val Glu Leu Glu Val Leu Gly 225 230 235 240 GAT ACA GAG GGC CTC AAT CTT TCG TTC TCA GCT GTC TGT AAC AAT GGC 768 Asp Thr Glu Gly Leu Asn Leu Ser Phe Ser Ala Val Cys Asn Asn Gly 245 250 255 ACT CTC TTC CCA CAC CAA AAG AAA TGC TTG CAC ATG AAA GTG GGA GAA 816 Thr Leu Phe Pro His Gln Lys Lys Cys Leu His Met Lys Val Gly Glu 260 265 270 ACA GCT TCA TTC AAT GTG ACT GTG AGT ATA CCA AAC TGT GAG AGA AAA 864 Thr Ala Ser Phe Asn Val Thr Val Ser Ile Pro Asn Cys Glu Arg Lys 275 280 285 AGC AGG CAT GTT ATC ATA AAG CCT GTG GGG CTG GGG GAC ACC CTG GAA 912 Ser Arg His Val Ile Ile Lys Pro Val Gly Leu Gly Asp Thr Leu Glu 290 295 300 ATC CTT GTC AGC CCA GAA TGC AGC TGC GAT TGT CAG AAA GAA GTG GAA 960 Ile Leu Val Ser Pro Glu Cys Ser Cys Asp Cys Gln Lys Glu Val Glu 305 310 315 320 GTG AAC AGC TCC AAA TGC CAC AAT GGG AAC GGC TCC TAC CAG TGT GGG 1008 Val Asn Ser Ser Lys Cys His Asn Gly Asn Gly Ser Tyr Gln Cys Gly 325 330 335 GTG TGT GCC TGT AAC CCA GGC CAC ATG GGC CCT CAC TGC GAG TGT GGT 1056 Val Cys Ala Cys Asn Pro Gly His Met Gly Pro His Cys Glu Cys Gly 340 345 350 GAG GAC ACG CTG AGC ACA GAT TCC TGC AAG GAG ACC CCA GAC CAT CCC 1104 Glu Asp Thr Leu Ser Thr Asp Ser Cys Lys Glu Thr Pro Asp His Pro 355 360 365 TCG TGC AGC GGA AGG GGT GAC TGC TAC TGT GGG CAG TGC ATC TGC CAC 1152 Ser Cys Ser Gly Arg Gly Asp Cys Tyr Cys Gly Gln Cys Ile Cys His 370 375 380 TTG TCT CCC TAT GGA AAC ATT TAT GGA CCT TAC TGC CAG TGT GAC AAT 1200 Leu Ser Pro Tyr Gly Asn Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn 385 390 395 400 TTC TCC TGT GTG AGG CAC AAA GGG CTG CTC TGT GGA GAT AAC GGA GAC 1248 Phe Ser Cys Val Arg His Lys Gly Leu Leu Cys Gly Asp Asn Gly Asp 405 410 415 TGT GAA TGT GGG GAA TGC GTG TGC AGG AGT GGT TGG ACC GGA GAG TAC 1296 Cys Glu Cys Gly Glu Cys Val Cys Arg Ser Gly Trp Thr Gly Glu Tyr 420 425 430 TGC AAC TGT ACC ACC AGC ACA GAC ACC TGC ATC TCC GAA GAC GGC ACG 1344 Cys Asn Cys Thr Thr Ser Thr Asp Thr Cys Ile Ser Glu Asp Gly Thr 435 440 445 CTC TGC AGC GGG CGC GGG GAC TGC GTC TGT GGC AAG TGT GTC TGC ACG 1392 Leu Cys Ser Gly Arg Gly Asp Cys Val Cys Gly Lys Cys Val Cys Thr 450 455 460 AAC CCT GGA GCC TCG GGA CCC ACC TGT GAA CGA TGT CCT ACC TGT AGT 1440 Asn Pro Gly Ala Ser Gly Pro Thr Cys Glu Arg Cys Pro Thr Cys Ser 465 470 475 480 GAC CCC TGT AAC TCT AAA CGG AGC TGC ATT GAA TGC CAC CTG TCT GCA 1488 Asp Pro Cys Asn Ser Lys Arg Ser Cys Ile Glu Cys His Leu Ser Ala 485 490 495 GAT GGT CAG CCT GGA GAA GAA TGT GTG GAC AAA TGC AAA CTA GCA GGT 1536 Asp Gly Gln Pro Gly Glu Glu Cys Val Asp Lys Cys Lys Leu Ala Gly 500 505 510 GTG ACC ATC AGC AAA GAA GCA GAT TTC TCA AAG GAT AGT TCT GTT TCC 1584 Val Thr Ile Ser Lys Glu Ala Asp Phe Ser Lys Asp Ser Ser Val Ser 515 520 525 TGC TCC CTG CAA GGA GAA AAT GAA TGT CTT ATT ACA TTC CTA ATA AGT 1632 Cys Ser Leu Gln Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu Ile Ser 530 535 540 ACA GAT AAT GAG GGA AAA ACC ATC ATT CAC AAC ATC AGT GAA AAA GAC 1680 Thr Asp Asn Glu Gly Lys Thr Ile Ile His Asn Ile Ser Glu Lys Asp 545 550 555 560 TGC CCC AAA CCT CCA AAT ATT CCT ATG ATC ATG TTG GGG GTT TCA CTG 1728 Cys Pro Lys Pro Pro Asn Ile Pro Met Ile Met Leu Gly Val Ser Leu 565 570 575 GCT A 1732 Ala 577 amino acids amino acid linear protein 29 Ser Ala Ser Met Asp Asp Asp Leu Asn Thr Ile Lys Glu Leu Gly Ser 1 5 10 15 Leu Leu Ser Lys Glu Met Ser Lys Leu Thr Ser Asn Phe Arg Leu Gly 20 25 30 Phe Gly Ser Phe Val Glu Lys Pro Val Ser Pro Phe Met Lys Thr Thr 35 40 45 Pro Glu Glu Ile Ala Asn Pro Cys Ser Ser Ile Pro Tyr Ile Cys Leu 50 55 60 Pro Thr Phe Gly Phe Lys His Ile Leu Pro Leu Thr Asn Asp Ala Glu 65 70 75 80 Arg Phe Asn Glu Ile Val Lys Lys Gln Lys Ile Ser Ala Asn Ile Asp 85 90 95 Asn Pro Glu Gly Gly Phe Asp Ala Ile Met Gln Ala Ala Val Cys Lys 100 105 110 Glu Lys Ile Gly Trp Arg Asn Asp Ser Leu His Leu Leu Val Phe Val 115 120 125 Ser Asp Ala Asp Ser His Phe Gly Met Asp Ser Lys Leu Ala Gly Ile 130 135 140 Val Ile Pro Asn Asp Gly Leu Cys His Leu Asp Ser Lys Asn Glu Tyr 145 150 155 160 Ser Met Ser Thr Val Met Glu Tyr Pro Thr Ile Gly Gln Leu Ile Asp 165 170 175 Lys Val Val Gln Asn Asn Val Leu Leu Ile Phe Ala Val Thr Gln Glu 180 185 190 Gln Val Pro Leu Tyr Glu Asn Tyr Ala Lys Leu Ile Pro Gly Ala Thr 195 200 205 Val Gly Leu Leu His Lys Asp Ser Gly Asn Ile Leu Gln Leu Ile Ile 210 215 220 Ser Ala Tyr Glu Glu Leu Arg Ser Glu Val Glu Leu Glu Val Leu Gly 225 230 235 240 Asp Thr Glu Gly Leu Asn Leu Ser Phe Ser Ala Val Cys Asn Asn Gly 245 250 255 Thr Leu Phe Pro His Gln Lys Lys Cys Leu His Met Lys Val Gly Glu 260 265 270 Thr Ala Ser Phe Asn Val Thr Val Ser Ile Pro Asn Cys Glu Arg Lys 275 280 285 Ser Arg His Val Ile Ile Lys Pro Val Gly Leu Gly Asp Thr Leu Glu 290 295 300 Ile Leu Val Ser Pro Glu Cys Ser Cys Asp Cys Gln Lys Glu Val Glu 305 310 315 320 Val Asn Ser Ser Lys Cys His Asn Gly Asn Gly Ser Tyr Gln Cys Gly 325 330 335 Val Cys Ala Cys Asn Pro Gly His Met Gly Pro His Cys Glu Cys Gly 340 345 350 Glu Asp Thr Leu Ser Thr Asp Ser Cys Lys Glu Thr Pro Asp His Pro 355 360 365 Ser Cys Ser Gly Arg Gly Asp Cys Tyr Cys Gly Gln Cys Ile Cys His 370 375 380 Leu Ser Pro Tyr Gly Asn Ile Tyr Gly Pro Tyr Cys Gln Cys Asp Asn 385 390 395 400 Phe Ser Cys Val Arg His Lys Gly Leu Leu Cys Gly Asp Asn Gly Asp 405 410 415 Cys Glu Cys Gly Glu Cys Val Cys Arg Ser Gly Trp Thr Gly Glu Tyr 420 425 430 Cys Asn Cys Thr Thr Ser Thr Asp Thr Cys Ile Ser Glu Asp Gly Thr 435 440 445 Leu Cys Ser Gly Arg Gly Asp Cys Val Cys Gly Lys Cys Val Cys Thr 450 455 460 Asn Pro Gly Ala Ser Gly Pro Thr Cys Glu Arg Cys Pro Thr Cys Ser 465 470 475 480 Asp Pro Cys Asn Ser Lys Arg Ser Cys Ile Glu Cys His Leu Ser Ala 485 490 495 Asp Gly Gln Pro Gly Glu Glu Cys Val Asp Lys Cys Lys Leu Ala Gly 500 505 510 Val Thr Ile Ser Lys Glu Ala Asp Phe Ser Lys Asp Ser Ser Val Ser 515 520 525 Cys Ser Leu Gln Gly Glu Asn Glu Cys Leu Ile Thr Phe Leu Ile Ser 530 535 540 Thr Asp Asn Glu Gly Lys Thr Ile Ile His Asn Ile Ser Glu Lys Asp 545 550 555 560 Cys Pro Lys Pro Pro Asn Ile Pro Met Ile Met Leu Gly Val Ser Leu 565 570 575 Ala 798 amino acids amino acid single linear 30 Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys 1 5 10 15 Cys Val Phe Ala Gln Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala 20 25 30 Lys Ser Cys Gly Glu Cys Ile Gln Ala Gly Pro Asn Cys Gly Trp Cys 35 40 45 Thr Asn Ser Thr Phe Phe Gln Glu Gly Met Pro Thr Ser Ala Arg Cys 50 55 60 Asp Asp Leu Glu Ala Leu Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile 65 70 75 80 Glu Asn Pro Arg Gly Ser Lys Asp Ile Lys Lys Asn Lys Asn Val Thr 85 90 95 Asn Arg Ser Lys Gly Thr Ala Glu Lys Leu Lys Pro Glu Asp Ile His 100 105 110 Gln Ile Gln Pro Gln Gln Leu Val Leu Arg Leu Arg Ser Gly Glu Pro 115 120 125 Gln Thr Phe Thr Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp 130 135 140 Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu 145 150 155 160 Asn Val Lys Ser Leu Gly Thr Asp Leu Met Asn Glu Met Arg Arg Ile 165 170 175 Thr Ser Asp Phe Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr Val 180 185 190 Met Pro Tyr Ile Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr 195 200 205 Ser Glu Gln Asn Cys Thr Thr Pro Phe Ser Tyr Lys Asn Val Leu Ser 210 215 220 Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Leu Val Gly Lys Gln Arg 225 230 235 240 Ile Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met 245 250 255 Gln Val Ala Val Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg 260 265 270 Leu Leu Val Phe Ser Thr Asp Ala Gly Phe His Phe Ala Gly Asp Gly 275 280 285 Lys Leu Gly Gly Ile Val Leu Pro Asn Asp Gly Gln Cys His Leu Glu 290 295 300 Asn Asn Met Tyr Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala 305 310 315 320 His Leu Val Gln Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala 325 330 335 Val Thr Glu Glu Phe Gln Pro Val Tyr Lys Glu Leu Lys Asn Leu Ile 340 345 350 Pro Lys Ser Ala Val Gly Thr Leu Ser Ala Asn Ser Ser Asn Val Ile 355 360 365 Gln Leu Ile Ile Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu 370 375 380 Glu Asn Gly Lys Leu Ser Glu Gly Val Thr Ile Ser Tyr Lys Ser Tyr 385 390 395 400 Cys Lys Asn Gly Val Asn Gly Thr Gly Glu Asn Gly Arg Lys Cys Ser 405 410 415 Asn Ile Ser Ile Gly Asp Glu Val Gln Phe Glu Ile Ser Ile Thr Ser 420 425 430 Asn Lys Cys Pro Lys Lys Asp Ser Asp Ser Phe Lys Ile Arg Pro Leu 435 440 445 Gly Phe Thr Glu Glu Val Glu Val Ile Leu Gln Tyr Ile Cys Glu Cys 450 455 460 Glu Cys Gln Ser Glu Gly Ile Pro Glu Ser Pro Lys Cys His Glu Gly 465 470 475 480 Asn Gly Thr Phe Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg Val 485 490 495 Gly Arg His Cys Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met 500 505 510 Asp Ala Tyr Cys Arg Lys Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn 515 520 525 Gly Glu Cys Val Cys Gly Gln Cys Val Cys Arg Lys Arg Asp Asn Thr 530 535 540 Asn Glu Ile Tyr Ser Gly Lys Phe Cys Glu Cys Asp Asn Phe Asn Cys 545 550 555 560 Asp Arg Ser Asn Gly Leu Ile Cys Gly Gly Asn Gly Val Cys Lys Cys 565 570 575 Arg Val Cys Glu Cys Asn Pro Asn Tyr Thr Gly Ser Ala Cys Asp Cys 580 585 590 Ser Leu Asp Thr Ser Thr Cys Glu Ala Ser Asn Gly Gln Ile Cys Asn 595 600 605 Gly Arg Gly Ile Cys Glu Cys Gly Val Cys Lys Cys Thr Asp Pro Lys 610 615 620 Phe Gln Gly Gln Thr Cys Glu Met Cys Gln Thr Cys Leu Gly Val Cys 625 630 635 640 Ala Glu His Lys Glu Cys Val Gln Cys Arg Ala Phe Asn Lys Gly Glu 645 650 655 Lys Lys Asp Thr Cys Thr Gln Glu Cys Ser Tyr Phe Asn Ile Thr Lys 660 665 670 Val Glu Ser Arg Asp Lys Leu Pro Gln Pro Val Gln Pro Asp Pro Val 675 680 685 Ser His Cys Lys Glu Lys Asp Val Asp Asp Cys Trp Phe Tyr Phe Thr 690 695 700 Tyr Ser Val Asn Gly Asn Asn Glu Val Met Val His Val Val Glu Asn 705 710 715 720 Pro Glu Cys Pro Thr Gly Pro Asp Ile Ile Pro Ile Val Ala Gly Val 725 730 735 Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys 740 745 750 Leu Leu Met Ile Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys 755 760 765 Glu Lys Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Tyr Lys 770 775 780 Ser Ala Val Thr Thr Val Val Asn Pro Lys Tyr Glu Gly Lys 785 790 795 769 amino acids amino acid single linear 31 Met Leu Gly Leu Arg Pro Pro Leu Leu Ala Leu Val Gly Leu Leu Se 1 5 10 15 Leu Gly Cys Val Leu Ser Gln Glu Cys Thr Lys Phe Lys Val Ser Se 20 25 30 Cys Arg Glu Cys Ile Glu Ser Gly Pro Gly Cys Thr Trp Cys Gln Ly 35 40 45 Leu Asn Phe Thr Gly Pro Gly Asp Pro Asp Ser Ile Arg Cys Asp Th 50 55 60 Arg Pro Gln Leu Leu Met Arg Gly Cys Ala Ala Asp Asp Ile Met As 65 70 75 80 Pro Thr Ser Leu Ala Glu Thr Gln Glu Asp His Asn Gly Gly Gln Ly 85 90 95 Gln Leu Ser Pro Gln Lys Val Thr Leu Tyr Leu Arg Pro Gly Gln Al 100 105 110 Ala Ala Phe Asn Val Thr Phe Arg Arg Ala Lys Gly Tyr Pro Ile As 115 120 125 Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Leu Asp Asp Leu Ar 130 135 140 Asn Val Lys Lys Leu Gly Gly Asp Leu Leu Arg Ala Leu Asn Glu Il 145 150 155 160 Thr Glu Ser Gly Arg Ile Gly Phe Gly Ser Phe Val Asp Lys Thr Va 165 170 175 Leu Pro Phe Val Asn Thr His Pro Asp Lys Leu Arg Asn Pro Cys Pr 180 185 190 Asn Lys Glu Lys Glu Cys Gln Pro Pro Phe Ala Phe Arg His Val Le 195 200 205 Lys Leu Thr Asn Asn Ser Asn Gln Phe Gln Thr Glu Val Gly Lys Gl 210 215 220 Leu Ile Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Leu Asp Ala Me 225 230 235 240 Met Gln Val Ala Ala Cys Pro Glu Glu Ile Gly Trp Arg Asn Val Th 245 250 255 Arg Leu Leu Val Phe Ala Thr Asp Asp Gly Phe His Phe Ala Gly As 260 265 270 Gly Lys Leu Gly Ala Ile Leu Thr Pro Asn Asp Gly Arg Cys His Le 275 280 285 Glu Asp Asn Leu Tyr Lys Arg Ser Asn Glu Phe Asp Tyr Pro Ser Va 290 295 300 Gly Gln Leu Ala His Lys Leu Ala Glu Asn Asn Ile Gln Pro Ile Ph 305 310 315 320 Ala Val Thr Ser Arg Met Val Lys Thr Tyr Glu Lys Leu Thr Glu Il 325 330 335 Ile Pro Lys Ser Ala Val Gly Glu Leu Ser Glu Asp Ser Ser Asn Va 340 345 350 Val His Leu Ile Lys Asn Ala Tyr Asn Lys Leu Ser Ser Arg Val Ph 355 360 365 Leu Asp His Asn Ala Leu Pro Asp Thr Leu Lys Val Thr Tyr Asp Se 370 375 380 Phe Cys Ser Asn Gly Val Thr His Arg Asn Gln Pro Arg Gly Asp Cy 385 390 395 400 Asp Gly Val Gln Ile Asn Val Pro Ile Thr Phe Gln Val Lys Val Th 405 410 415 Ala Thr Glu Cys Ile Gln Glu Gln Ser Phe Val Ile Arg Ala Leu Gl 420 425 430 Phe Thr Asp Ile Val Thr Val Gln Val Leu Pro Gln Cys Glu Cys Ar 435 440 445 Cys Arg Asp Gln Ser Arg Asp Arg Ser Leu Cys His Gly Lys Gly Ph 450 455 460 Leu Glu Cys Gly Ile Cys Arg Cys Asp Thr Gly Tyr Ile Gly Lys As 465 470 475 480 Cys Glu Cys Gln Thr Gln Gly Arg Ser Ser Gln Glu Leu Glu Gly Se 485 490 495 Cys Arg Lys Asp Asn Asn Ser Ile Ile Cys Ser Gly Leu Gly Asp Cy 500 505 510 Val Cys Gly Gln Cys Leu Cys His Thr Ser Asp Val Pro Gly Lys Le 515 520 525 Ile Tyr Gly Gln Tyr Cys Glu Cys Asp Thr Ile Asn Cys Glu Arg Ty 530 535 540 Asn Gly Gln Val Cys Gly Gly Pro Gly Arg Gly Leu Cys Phe Cys Gl 545 550 555 560 Lys Cys Arg Cys His Pro Gly Phe Glu Gly Ser Ala Cys Gln Cys Gl 565 570 575 Arg Thr Thr Glu Gly Cys Leu Asn Pro Arg Arg Val Glu Cys Ser Gl 580 585 590 Arg Gly Arg Cys Arg Cys Asn Val Cys Glu Cys His Ser Gly Tyr Gl 595 600 605 Leu Pro Leu Cys Gln Glu Cys Pro Gly Cys Pro Ser Pro Cys Gly Ly 610 615 620 Tyr Ile Ser Cys Ala Glu Cys Leu Lys Phe Glu Lys Gly Pro Phe Gl 625 630 635 640 Lys Asn Cys Ser Ala Ala Cys Pro Gly Leu Gln Leu Ser Asn Asn Pr 645 650 655 Val Lys Gly Arg Thr Cys Lys Glu Arg Asp Ser Glu Gly Cys Trp Va 660 665 670 Ala Tyr Thr Leu Glu Gln Gln Asp Gly Met Asp Arg Tyr Leu Ile Ty 675 680 685 Val Asp Glu Ser Arg Glu Cys Val Ala Gly Pro Asn Ile Ala Ala Il 690 695 700 Val Gly Gly Thr Val Ala Gly Ile Val Leu Ile Gly Ile Leu Leu Le 705 710 715 720 Val Ile Trp Lys Ala Leu Ile His Leu Ser Asp Leu Arg Glu Tyr Ar 725 730 735 Arg Phe Glu Lys Glu Lys Leu Lys Ser Gln Trp Asn Asn Asp Asn Pr 740 745 750 Leu Phe Lys Ser Ala Thr Thr Thr Val Met Asn Pro Lys Phe Ala Gl 755 760 765 Ser 788 amino acids amino acid single linear 32 Met Arg Ala Arg Pro Arg Pro Arg Pro Leu Trp Val Thr Val Leu Ala 1 5 10 15 Leu Gly Ala Leu Ala Gly Val Gly Val Gly Gly Pro Asn Ile Cys Thr 20 25 30 Thr Arg Gly Val Ser Ser Cys Gln Gln Cys Leu Ala Val Ser Pro Met 35 40 45 Cys Ala Trp Cys Ser Asp Glu Ala Leu Pro Leu Gly Ser Pro Arg Cys 50 55 60 Asp Leu Lys Glu Asn Leu Leu Lys Asp Asn Cys Ala Pro Glu Ser Ile 65 70 75 80 Glu Phe Pro Val Ser Glu Ala Arg Val Leu Glu Asp Arg Pro Leu Ser 85 90 95 Asp Lys Gly Ser Gly Asp Ser Ser Gln Val Thr Gln Val Ser Pro Gln 100 105 110 Arg Ile Ala Leu Arg Leu Arg Pro Asp Asp Ser Lys Asn Phe Ser Ile 115 120 125 Gln Val Arg Gln Val Glu Asp Tyr Pro Val Asp Ile Tyr Tyr Leu Met 130 135 140 Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Trp Ser Ile Gln Asn Leu 145 150 155 160 Gly Thr Lys Leu Ala Thr Gln Met Arg Lys Leu Thr Ser Asn Leu Arg 165 170 175 Ile Gly Phe Gly Ala Phe Val Asp Lys Pro Val Ser Pro Tyr Met Tyr 180 185 190 Ile Ser Pro Pro Glu Ala Leu Glu Asn Pro Cys Tyr Asp Met Lys Thr 195 200 205 Thr Cys Leu Pro Met Phe Gly Tyr Lys His Val Leu Thr Leu Thr Asp 210 215 220 Gln Val Thr Arg Phe Asn Glu Glu Val Lys Lys Gln Ser Val Ser Arg 225 230 235 240 Asn Arg Asp Ala Pro Glu Gly Gly Phe Asp Ala Ile Met Gln Ala Thr 245 250 255 Val Cys Asp Glu Lys Ile Gly Trp Arg Asn Asp Ala Ser His Leu Leu 260 265 270 Val Phe Thr Thr Asp Ala Lys Thr His Ile Ala Leu Asp Gly Arg Leu 275 280 285 Ala Gly Ile Val Gln Pro Asn Asp Gly Gln Cys His Val Gly Ser Asp 290 295 300 Asn His Tyr Ser Ala Ser Thr Thr Met Asp Tyr Pro Ser Leu Gly Leu 305 310 315 320 Met Thr Glu Lys Leu Ser Gln Lys Asn Ile Asn Leu Ile Phe Ala Val 325 330 335 Thr Glu Asn Val Val Asn Leu Tyr Gln Asn Tyr Ser Glu Leu Ile Pro 340 345 350 Gly Thr Thr Val Gly Val Leu Ser Met Asp Ser Ser Asn Val Leu Gln 355 360 365 Leu Ile Val Asp Ala Tyr Gly Lys Ile Arg Ser Lys Val Glu Leu Glu 370 375 380 Val Arg Asp Leu Pro Glu Glu Leu Ser Leu Ser Phe Asn Ala Thr Cys 385 390 395 400 Leu Asn Asn Glu Val Ile Pro Gly Leu Lys Ser Cys Met Gly Leu Lys 405 410 415 Ile Gly Asp Thr Val Ser Phe Ser Ile Glu Ala Lys Val Arg Gly Cys 420 425 430 Pro Gln Glu Lys Glu Lys Ser Phe Thr Ile Lys Pro Val Gly Phe Lys 435 440 445 Asp Ser Leu Ile Val Gln Val Thr Phe Asp Cys Asp Cys Ala Cys Gln 450 455 460 Ala Gln Ala Glu Pro Asn Ser His Arg Cys Asn Asn Gly Asn Gly Thr 465 470 475 480 Phe Glu Cys Gly Val Cys Arg Cys Gly Pro Gly Trp Leu Gly Ser Gln 485 490 495 Cys Glu Cys Ser Glu Glu Asp Tyr Arg Pro Ser Gln Gln Asp Glu Cys 500 505 510 Ser Pro Arg Glu Gly Gln Pro Val Cys Ser Gln Arg Gly Glu Cys Leu 515 520 525 Cys Gly Gln Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly 530 535 540 Lys Tyr Cys Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu 545 550 555 560 Met Cys Ser Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp 565 570 575 Ser Asp Trp Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Arg Thr Asp Thr 580 585 590 Cys Met Ser Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu 595 600 605 Cys Gly Ser Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr Cys 610 615 620 Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys Thr Phe Lys Lys Glu Cys 625 630 635 640 Val Glu Cys Lys Lys Phe Asp Arg Glu Pro Tyr Met Thr Glu Asn Thr 645 650 655 Cys Asn Arg Tyr Cys Arg Asp Glu Ile Glu Ser Val Lys Glu Leu Lys 660 665 670 Asp Thr Gly Lys Asp Ala Val Asn Cys Thr Tyr Lys Asn Glu Asp Asp 675 680 685 Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp Ser Ser Gly Lys Ser Ile 690 695 700 Leu Tyr Val Val Glu Glu Pro Glu Cys Pro Lys Gly Pro Asp Ile Leu 705 710 715 720 Val Val Leu Leu Ser Val Met Gly Ala Ile Leu Leu Ile Gly Leu Ala 725 730 735 Ala Leu Leu Ile Trp Lys Leu Leu Ile Thr Ile His Asp Arg Lys Glu 740 745 750 Phe Ala Lys Phe Glu Glu Glu Arg Ala Arg Ala Lys Trp Asp Thr Ala 755 760 765 Asn Asn Pro Leu Tyr Lys Glu Ala Thr Ser Thr Phe Thr Asn Ile Thr 770 775 780 Tyr Arg Gly Thr 785 846 amino acids amino acid single linear 33 Met Ile Leu Glu Arg Asn Arg Arg Cys Gln Leu Ala Leu Leu Met Ile 1 5 10 15 Ala Met Leu Ala Ala Ile Ala Ala Gln Thr Asn Ala Gln Lys Ala Ala 20 25 30 Lys Leu Thr Ala Val Ser Thr Cys Ala Ser Lys Glu Lys Cys His Thr 35 40 45 Cys Ile Gln Thr Glu Gly Cys Ala Trp Cys Met Gln Pro Asp Phe Lys 50 55 60 Gly Gln Ser Arg Cys Tyr Gln Asn Thr Ser Ser Leu Cys Pro Glu Glu 65 70 75 80 Phe Ala Tyr Ser Pro Ile Thr Val Glu Gln Ile Leu Val Asn Asn Lys 85 90 95 Leu Thr Asn Gln Tyr Lys Ala Glu Leu Ala Ala Gly Gly Gly Gly Gly 100 105 110 Ala Met Ser Gly Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser 115 120 125 Ser Ser Phe Tyr Ser Gln Ser Ser Ser Gly Ser Ser Ser Ala Ser Gly 130 135 140 Tyr Glu Glu Tyr Ser Ala Gly Glu Ile Val Gln Ile Gln Pro Gln Ser 145 150 155 160 Met Arg Leu Ala Leu Arg Val Asn Glu Lys His Asn Ile Lys Ile Ser 165 170 175 Tyr Ser Gln Ala Glu Gly Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp 180 185 190 Leu Ser Lys Ser Met Glu Asp Asp Lys Ala Lys Leu Ser Thr Leu Gly 195 200 205 Asp Lys Leu Ser Glu Thr Met Lys Arg Ile Thr Asn Asn Phe His Leu 210 215 220 Gly Phe Gly Ser Phe Val Asp Lys Val Leu Met Pro Tyr Val Ser Thr 225 230 235 240 Ile Pro Lys Lys Leu Glu His Pro Cys Glu Asn Cys Lys Ala Pro Tyr 245 250 255 Gly Tyr Gln Asn His Met Pro Leu Asn Asn Asn Thr Glu Ser Phe Ser 260 265 270 Asn Glu Val Lys Asn Ala Thr Val Ser Gly Asn Leu Asp Ala Pro Glu 275 280 285 Gly Gly Phe Asp Ala Ile Met Gln Ala Ile Ala Cys Arg Ser Gln Ile 290 295 300 Gly Trp Arg Glu Gln Ala Arg Arg Leu Leu Val Phe Ser Thr Asp Ala 305 310 315 320 Gly Phe His Tyr Ala Gly Asp Gly Lys Leu Gly Gly Val Ile Ala Pro 325 330 335 Asn Asp Gly Glu Cys His Leu Ser Pro Lys Gly Glu Tyr Thr His Ser 340 345 350 Thr Leu Gln Asp Tyr Pro Ser Ile Ser Gln Ile Asn Gln Lys Val Lys 355 360 365 Asp Asn Ala Ile Asn Ile Ile Phe Ala Val Thr Ala Ser Gln Leu Ser 370 375 380 Val Tyr Glu Lys Leu Val Glu His Ile Gln Gly Ser Ser Ala Ala Lys 385 390 395 400 Leu Asp Asn Asp Ser Ser Asn Val Val Glu Leu Val Lys Glu Glu Tyr 405 410 415 Arg Lys Ile Ser Ser Ser Val Glu Met Lys Asp Asn Ala Thr Gly Asp 420 425 430 Val Lys Ile Thr Tyr Phe Ser Ser Cys Leu Ser Asn Gly Pro Glu Val 435 440 445 Gln Thr Ser Lys Cys Asp Asn Leu Lys Glu Gly Gln Gln Val Ser Phe 450 455 460 Thr Ala Gln Ile Gln Leu Leu Lys Cys Pro Glu Asp Pro Arg Asp Trp 465 470 475 480 Thr Gln Thr Ile His Ile Ser Pro Val Gly Ile Asn Glu Val Met Gln 485 490 495 Ile Gln Leu Thr Met Leu Cys Ser Cys Pro Cys Glu Asn Pro Gly Ser 500 505 510 Ile Gly Tyr Gln Val Gln Ala Asn Ser Cys Ser Gly His Gly Thr Ser 515 520 525 Met Cys Gly Ile Cys Asn Cys Asp Asp Ser Tyr Phe Gly Asn Lys Cys 530 535 540 Glu Cys Ser Ala Thr Asp Leu Thr Ser Lys Phe Ala Asn Asp Thr Ser 545 550 555 560 Cys Arg Ala Asp Ser Thr Ser Thr Thr Asp Cys Ser Gly Arg Gly His 565 570 575 Cys Cys Val Gly Ala Cys Glu Cys His Lys Arg Pro Asn Pro Ile Glu 580 585 590 Ile Ile Ser Gly Lys His Cys Glu Cys Asp Asn Phe Ser Cys Glu Arg 595 600 605 Asn Arg Asn Gln Leu Cys Ser Gly Pro Asp His Gly Thr Cys Glu Cys 610 615 620 Gly Arg Cys Lys Cys Lys Pro Gly Trp Thr Gly Ser Asn Cys Gly Cys 625 630 635 640 Gln Glu Ser Asn Asp Thr Cys Met Pro Pro Gly Gly Gly Glu Ile Cys 645 650 655 Ser Gly His Gly Thr Cys Glu Cys Gly Val Cys Lys Cys Thr Val Asn 660 665 670 Asp Gln Gly Arg Phe Ser Gly Arg His Cys Glu Lys Cys Pro Thr Cys 675 680 685 Ser Gly Arg Cys Gln Glu Leu Lys Asp Cys Val Gln Cys Gln Met Tyr 690 695 700 Lys Thr Gly Glu Leu Lys Asn Gly Asp Asp Cys Ala Arg Asn Cys Thr 705 710 715 720 Gln Phe Val Pro Val Gly Val Glu Lys Val Glu Ile Asp Glu Thr Lys 725 730 735 Asp Glu Gln Met Cys Lys Phe Phe Asp Glu Asp Asp Cys Lys Phe Met 740 745 750 Phe Lys Tyr Ser Glu Gln Gly Glu Leu His Val Tyr Ala Gln Glu Asn 755 760 765 Lys Glu Cys Pro Ala Lys Val Phe Met Leu Gly Ile Val Met Gly Val 770 775 780 Ile Ala Ala Ile Val Leu Val Gly Leu Ala Ile Leu Leu Leu Trp Lys 785 790 795 800 Leu Leu Thr Thr Ile His Asp Arg Arg Glu Phe Ala Arg Phe Glu Lys 805 810 815 Glu Arg Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Tyr Lys 820 825 830 Gln Ala Thr Ser Thr Phe Lys Asn Pro Met Tyr Ala Gly Lys 835 840 845 282 base pairs nucleic acid double linear CDS 1..282 34 TGT GTT TGT AGG AAG AGG GAT AAT ACA AAT GAA ATT TAT TCT GGC AAA 48 Cys Val Cys Arg Lys Arg Asp Asn Thr Asn Glu Ile Tyr Ser Gly Lys 1 5 10 15 TTC TGC GAG TGT GAT AAT TTC AAC TGT GAT AGA TCC AAT GGC TTA ATT 96 Phe Cys Glu Cys Asp Asn Phe Asn Cys Asp Arg Ser Asn Gly Leu Ile 20 25 30 TGT GGA GGA AAT GGT GTT TGC AAG TGT CGT GTG TGT GAG TGC AAC CCC 144 Cys Gly Gly Asn Gly Val Cys Lys Cys Arg Val Cys Glu Cys Asn Pro 35 40 45 AAC TAC ACT GGC AGT GCA TGT GAC TGT TCT TTG GAT ACT AGT ACT TGT 192 Asn Tyr Thr Gly Ser Ala Cys Asp Cys Ser Leu Asp Thr Ser Thr Cys 50 55 60 GAA GCC AGC AAC GGA CAG ATC TGC AAT GGC CGG GGC ATC TGC GAG TGT 240 Glu Ala Ser Asn Gly Gln Ile Cys Asn Gly Arg Gly Ile Cys Glu Cys 65 70 75 80 GGT GTC TGT AAG TGT ACA GAT CCG AAG TTT CAA GGG CAA ACG 282 Gly Val Cys Lys Cys Thr Asp Pro Lys Phe Gln Gly Gln Thr 85 90 94 amino acids amino acid linear protein 35 Cys Val Cys Arg Lys Arg Asp Asn Thr Asn Glu Ile Tyr Ser Gly Lys 1 5 10 15 Phe Cys Glu Cys Asp Asn Phe Asn Cys Asp Arg Ser Asn Gly Leu Ile 20 25 30 Cys Gly Gly Asn Gly Val Cys Lys Cys Arg Val Cys Glu Cys Asn Pro 35 40 45 Asn Tyr Thr Gly Ser Ala Cys Asp Cys Ser Leu Asp Thr Ser Thr Cys 50 55 60 Glu Ala Ser Asn Gly Gln Ile Cys Asn Gly Arg Gly Ile Cys Glu Cys 65 70 75 80 Gly Val Cys Lys Cys Thr Asp Pro Lys Phe Gln Gly Gln Thr 85 90 282 base pairs nucleic acid double linear CDS 1..282 36 TGC GTG TGC AGG AAG AGG GAC AAC ACC AAC GAG ATC TAC TCG GGC AAA 48 Cys Val Cys Arg Lys Arg Asp Asn Thr Asn Glu Ile Tyr Ser Gly Lys 1 5 10 15 TTC TGC GAG TGC GAC AAC TTC AAC TGT GAT CGG TCC AAT GGC TTA ATC 96 Phe Cys Glu Cys Asp Asn Phe Asn Cys Asp Arg Ser Asn Gly Leu Ile 20 25 30 TGT GGA GGC AAT GGA GTG TGC CGG TGT CGT GTG TGC GAG TGC TTC CCC 144 Cys Gly Gly Asn Gly Val Cys Arg Cys Arg Val Cys Glu Cys Phe Pro 35 40 45 AAC TAC ACC GGC AGC GCC TGT GAC TGC TCT CTG GAC ACT GCG CCG TGC 192 Asn Tyr Thr Gly Ser Ala Cys Asp Cys Ser Leu Asp Thr Ala Pro Cys 50 55 60 CTG GCC ACC AAC GGG CAG ATC TGC AAT GGC CGG GGT GTG TGC GAG TGC 240 Leu Ala Thr Asn Gly Gln Ile Cys Asn Gly Arg Gly Val Cys Glu Cys 65 70 75 80 GGC GTG TGC AAG TGC ACG GAC CCC AAG TTC CAG GGG CAG ACC 282 Gly Val Cys Lys Cys Thr Asp Pro Lys Phe Gln Gly Gln Thr 85 90 94 amino acids amino acid linear protein 37 Cys Val Cys Arg Lys Arg Asp Asn Thr Asn Glu Ile Tyr Ser Gly Lys 1 5 10 15 Phe Cys Glu Cys Asp Asn Phe Asn Cys Asp Arg Ser Asn Gly Leu Ile 20 25 30 Cys Gly Gly Asn Gly Val Cys Arg Cys Arg Val Cys Glu Cys Phe Pro 35 40 45 Asn Tyr Thr Gly Ser Ala Cys Asp Cys Ser Leu Asp Thr Ala Pro Cys 50 55 60 Leu Ala Thr Asn Gly Gln Ile Cys Asn Gly Arg Gly Val Cys Glu Cys 65 70 75 80 Gly Val Cys Lys Cys Thr Asp Pro Lys Phe Gln Gly Gln Thr 85 90 276 base pairs nucleic acid double linear CDS 1..276 38 TGT GTC TGC CAC AGC AGT GAC TTT GGC AAG ATC ACG GGC AAG TAC TGC 48 Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly Lys Tyr Cys 1 5 10 15 GAG TGT GAC GAC TTC TCC TGT GTC CGC TAC AAG GGG GAG ATG TGC TCA 96 Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu Met Cys Ser 20 25 30 GGC CAT GGC CAG TGC AGC TGT GGG GAC TGC CTG TGT GAC TCC GAC TGG 144 Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp Ser Asp Trp 35 40 45 ACC GGC TAC TAC TGC AAC TGT ACC ACG CGT ACT GAC ACC TGC ATG TCC 192 Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Arg Thr Asp Thr Cys Met Ser 50 55 60 AGC AAT GGG CTG CTG TGC AGC GGC CGC GGC AAG TGT GAA TGT GGC AGC 240 Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu Cys Gly Ser 65 70 75 80 TGT GTC TGT ATC CAG CCG GGC TCC TAT GGG GAC ACC 276 Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr 85 90 92 amino acids amino acid linear protein 39 Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly Lys Tyr Cys 1 5 10 15 Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu Met Cys Ser 20 25 30 Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp Ser Asp Trp 35 40 45 Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Arg Thr Asp Thr Cys Met Ser 50 55 60 Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu Cys Gly Ser 65 70 75 80 Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr 85 90 276 base pairs nucleic acid double linear CDS 1..276 40 TGC TCC TGC CAC AGC GAT GAC TTT GGC AAG ATC ACG GGC AAG TAC TGT 48 Cys Ser Cys His Ser Asp Asp Phe Gly Lys Ile Thr Gly Lys Tyr Cys 1 5 10 15 GAG TGT GAT GAC TTC TCC TGT GTT CGC TAC AAA GGG GAG ATG TGC TCA 96 Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu Met Cys Ser 20 25 30 GGC CAT GGC CAG TGC AGC TGT GGG GAT TGC CTG TGT GAT TCT GAC TGG 144 Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp Ser Asp Trp 35 40 45 ACT GGC TAC TAC TGT AAC TGT ACC ACA CTC ACT GAC ACC TGC ATG TCC 192 Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Leu Thr Asp Thr Cys Met Ser 50 55 60 AGC AAC GGG CTG TTG TGC AGC GGC CGG GGC AAG TGT GAA TGT GGC AGT 240 Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu Cys Gly Ser 65 70 75 80 TGT GTC TGC ATC CAG CCG GGA TCT TAT GGG GAC ACT 276 Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr 85 90 92 amino acids amino acid linear protein 41 Cys Ser Cys His Ser Asp Asp Phe Gly Lys Ile Thr Gly Lys Tyr Cys 1 5 10 15 Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly Glu Met Cys Ser 20 25 30 Gly His Gly Gln Cys Ser Cys Gly Asp Cys Leu Cys Asp Ser Asp Trp 35 40 45 Thr Gly Tyr Tyr Cys Asn Cys Thr Thr Leu Thr Asp Thr Cys Met Ser 50 55 60 Ser Asn Gly Leu Leu Cys Ser Gly Arg Gly Lys Cys Glu Cys Gly Ser 65 70 75 80 Cys Val Cys Ile Gln Pro Gly Ser Tyr Gly Asp Thr 85 90 15 amino acids amino acid single linear 42 Arg Gly Ser Thr Ser Thr Phe Lys Asn Val Thr Tyr Lys His Arg 1 5 10 15 15 amino acids amino acid single linear 43 Tyr Lys His Arg Glu Lys Gln Lys Val Asp Leu Ser Thr Asp Cys 1 5 10 15 

We claim:
 1. A method of decreasing cell adhesion in cells expressing an α_(V)β₆ integrin, the method comprising binding the α_(V)β₆ integrin with an antibody specifically reactive with a β₆ integrin subunit as displayed in SEQ ID NO:27. 